Chem 431 Lab Manual w 2016

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CHEM 431 LAB MANUAL

TABLE OF CONTENTS

Safety Rules............ 1

Lab Equipment....... 3

The Format o f Formal Lab Reports ... 5

Automatic Pipettes: Instruction for Use . 6

Lab #1: Checking the Accuracy and Precision of Pipettes ... 10

Lab #2: Quanti tative Measurement of Proteins .... 14

Lab #3: Electrophoresis of Serum Proteins Using SDS-PAGE .... 25

Drying Polyacrylamide Gels in Cellophane. 33

Analyzing the gel using the Image J Software .. 35

Lab #4: Restriction Enzyme Analysis of Circular DNA ... 43

Lab #5: Isolation of an Enzyme: Acid Phosphatase 51

Lab #6: Characterization of Acid Phosphatase .... 65

Instruct ions for the oral presentation .. 67

Appendix I: Introduct ion to UV-Vis Spectrophotometers .. 68

Instruct ions for UV-visible Spectrophotometers (Beckman DU-520) .. 70

Appendix II: Linear Regression Analysis: Excel 2007 71

Appendix III: Analyzing Agarose Gel Electrophoresis of DNA ..... 74

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SAFETY

C W U Chemistry Department

Teaching Laboratory Safety Rules

Updated 8/12/2015 (over)

The Chemistry Department is committed to providing a safe environment for students and staff. However,laboratory safety is a mutual responsibility and requires full participation and cooperation of all involved -students, TAs and instructors. The following lab safety rules have been established for your protection as a

student in the Chemistry Department. These rules will be rigidly and impartially enforced. Noncompliancemay result in a grading penalty and/or dismissal from lab. A statement of agreement to compliance withthe lab rules is included on the lab check-in sheet and must be signed prior to beginning lab.

Personal Protection Safety Goggles must be worn at all times in active chemistry labs. This is the policy of Central Washington

University and also a state requirement. The goggles must be of the indirect vented type and must meet ANSIZ87.1 specifications. The bookstore has approved gogglessafety glasses are not acceptable.Do not remove the plastic vent covers from the goggles.

The safety of wearing contact lenses in chemical laboratories has been hotly debated over the last several years.Both the ACS and OSHA have issued statements indicating that contact lenses can be worn IF AND ONLY IFproper protective eyewear is also worn. The Chemistry Department recognizes that some eye conditions require

contacts for certain vision correction therapies. However, students who choose to wear contacts must recognize

the inherent risksthey are difficult to remove if chemicals get in the eye, they have a tendency to preventnatural eye fluids from removing contaminants, and sudden displacement can cause visual problems that create

additional hazards. Soft contact lenses are especially problematic because they can discolor and also absorbchemical vapors causing damage before the wearer is alerted to the problem. If you choose to wear contacts,

please tell your lab instructor and write Contact Lenses Wearer by your signature on your lab check-in form. Appropriate gloves will be provided when needed. Use of gloves is required for handling certain chemicals. Gloves

are veryexpensive. Do not change gloves needlessly. Do not wear gloves outside the lab where they can

contaminate doors and other surfaces contacted. Wash hands after removing gloves, before leaving the lab. Appropriate clothing is required. Your clothing is a barrier between your skin and chemicals. All skin from

shoulders across the collar bone to toes must be completely covered (excluding hands). Lab coats are required

during the teaching labs by everyone at all times. They can be purchased at the student book store oronline. WAC 296-800-160 Code of Federal Regulations, Title 29 CFR, Part 1910, Subpart 1

Shoes must be worn and must completely cover all parts of the feet. WAC 296-800-160 Code of FederalRegulations, Title 29 CFR, Part 1910, Subpart 1 Long hair must be tied back. Do not eat, drink (including sport bottles), or store food in the labs.

Smoking or use of other tobacco products is prohibited. Wash hands after working with chemicals. Never mouth pipetteuse a pipette bulb. Do not taste chemicals. Check odors only if instructed by your Instructor- by wafting gently toward your nose

with your hand. It is the recommendation of this department that all students of reproductive age, especially women who have

recently conceived or are anticipating conception during the quarter, discuss the course content and reagents withtheir physician if concerned about reproductive toxins.

For your safety, if you have a medical condition that results in seizures, blackouts, etc. (e.g., from epilepsy,

diabetes) please inform your instructor who will direct you to the department safety representative. Thisinformation will be kept confidential. If you wish to seek accommodations due to a disability or to address

functional limitations, please contact Disability Services at 509-963-2214.

General Lab Rules No horseplay or running. Keep aisles clear - push stools against the bench when not in use. Store backpacks in

cubicles provided. No music allowed in student labs. Radios (including IPods) and other entertainment devices are not permitted. Read all instructions carefully and plan your work ahead of time. Understand the experiment and if in doubt, ask. Unsupervised laboratory work is not permitted. Do not perform unauthorized experiments. No personal phone calls are allowed on lab phones - public phones are available on the first floor, east side.

Cell phones should be turned off while in lab.

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Updated 8/12/2015

Chemistry lab computers are for chemistry business only - no Internet surfing or checking email. Handle chemicals carefully. Note the colored labels affixed to the bottles. The Chemistry Department follows the

J.T. Baker Saf-T-Data tmlabeling system.Colors represent hazard classes as follows:

Orange Minimal hazard, general precautions

Blue ToxicYellow Reactive, unstable

Yellow Stripe Incompatible with other YellowsRed FlammableRed Stripe Incompatible with other Reds

White Corrosive, usually acidsWhite Stripe Incompatible with other Whites, usually bases

Treat chemicals with respect and understand the chemicals you are using. Material Safety Data Sheets (MSDSs)

are available at the MSDS Workstation outside Room 311 and online. Do not remove the MSDSs from the binders;bring the binders to the Chem office (Room 302) to request a copy.

Dispose of waste chemicals in the appropriate labeled waste containersnever dispose of chemicals down thedrain unless directed to do so by the lab instructor.

Leave the lab area clean. Put equipment and chemicals away, wipe off the bench top and clean the balance pans.

JUST IN CASE

In case of an emergency, contact your TA and instructor immediately and be prepared as follows:

SpillsAssess the situation and notify the instructor. Small low level hazard spills can be cleaned up with thehelp of the TA using the spill kit in the prep room. If the spill is flammable, shut gas off immediately. If anychemical is spilled on the skin or splashed in the eyes, remove all jewelry and contaminated clothing and flush theaffected area with water for 15 min.

Glass BreakageDO NOT HANDLE broken glass with bare hands, consult TA or instructor on how to clean-up.Put the broken glass in the Broken Glass Containernot in the trashcan. Fill out a breakage slip and place it thebox in the prep room. The TA will use the mercury spill kit to clean up thermometersnever put them in thetrashcan.

Personal Injuryall injuries and near misses must be reported.

Minor injuries may be treated as follow:CutsRinse with water. Band-Aids are available from your TA.Thermal BurnsFlush with cold water. Do not cover, Report to your instructor/ TA immediately.Chemical BurnsFlush for 15 min. using sink, shower, or eyewash, Report to your instructor/ TA immediately.

Fill out a student accident report (forms located in First Aid drawer) and return signed form to the ChemistryStockroom (across from SCI 315).

Power OutageAwait instructions. If power is not restored in 15 min., your TA will help you to beginshutting down the lab. Put all chemicals away. Turn off gas and electrical equipment. Pull hood sashes down.

Fire Alarm SoundsIndicates imminent danger. Close chemical containers, shut off gas and electricity, exitfrom labs down the stairwelldo not use elevators. Your instructor will provide specific information concerning theremainder of the lab and re-entry into the building. Faculty, staff and TAs will assemble on the lawn on the North

side of the Japanese Garden. DO NOT CLEAN UP OR PUT THINGS AWAYEVACUATE IMMEDIATELY!

If you have any questions, the following people are your safety resources:- your instructor- the Chemistry Instructor

- the Chemistry Stockroom ManagerTony BrownSCI 303, 963-1303- the Chemistry Department Safety RepresentativeIan SeilerSCI 315, 509-607-1620- the campus Industrial Hygienist James Hudson963-2338- the campus Environmental Health & Safety OfficerRon Munson963-2252.

For additional department safety information visit the department safety web page athttp://www.cwu.edu/chemistry/laboratory-safety

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3

Lab Equipment

Beakers Wash bottles Three-prong clamp Clamp holder

Graduated cylinder Erlenmeyer flask Filter flask Buchner funnel

Polypropylene funnel Safety bulbs Micropipette

Microspatula Scoop spatula Mohr pipet

Spin bar Rubber policeman Thermometer

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Test tube Test tube brush Glass rod Test tube clamp Watch glass

Pipette tip holder Neoprene adapters Test tube rack Microfuge tube rack

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5

The Format of Formal Lab Reports

Two experiments will be described in a formal report organized in a research publication format.During the laboratory period data and observations must be recorded in a carbonless copy

laboratory notebook (purchased at the University Bookstore, e.g., Saunders Publishing). Writesimply and concisely in the passive or third person voice. In your report be sure to includeanswers to questions and any information or calculations specified in the lab book for eachexperiment. Always identify any unknown by number or letter. While you will work with apartner at times, each lab report must be written up individually. Each lab report must containthe following six parts presented in the order listed below:

1. Titleof the experiment and Nameof the author (lab partners names in parentheses).

2. Introduction:Clearly state the purpose of the experiment. State the question to beanswered or investigated and any background needed to introduce the subject. Statethe chemical principlesinvolved in the techniques being used.

3. Methods and Materials: If you followed directions exactly, this may be stated byreferencing the handout sheets. However, if changes were made in any materials orprocedures you must specify these in detail.

4. Results: Datamust be presented in tables or figures that are numbered and ti t led. Thetables and figures should be formatted in a manner similar to what is found in the peer-reviewed literature. Provide a sample calculat ionif a series of the same calculation isused, but list the series of calculated results in the table or figure. All numerical datamu st have units. Tables require column titles with units. Figures must have axeslabeled with units. Absorbance values and wavelengths at which the absorbance wasmeasured must be clearly stated on figures. A brief statement or summary of the results

observed must also be included in this section. Where multiple determinations aremade, means and standard deviations of the mean must be reported.

5. Discussion and Conclusions: State and discuss the significance of your observations.Were your observations surprising? Explain any deviations from expected results. Stateyour confidence in your conclusions based on the precision and accuracy of your dataand possible sources of error.

6. References and Appendix: Cite three peer-reviewed papers from the literature that isrelevant to the lab. Yellow pages of your laboratory notebook must be turned in withyour report showing the observations and calculations from the laboratory.

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Automatic Pipettes: Instructions for Use

You have three micropipettes that you will use for dispensing precise volumes of liquids.

It is crucial that you learn how to adjust and use these pipettes properly, as your results

will depend upon it. These pipettes are air displacement pipettes and work by creating avacuum that draws up a specified volume of liquid. Gaining proficiency with pipettes is

one of the most important skills for biochemists and your success in this course will

depend on your ability to pipette accurately. For more information on how this type of

pipettes work, you can follow this link. The three pipettes that you have in your drawer

dispense volumes within discrete ranges:

100 1000 L called the p1000 pipette



Pipettes tend to be more accurate towards the top of their ranges. Therefore, although

you can use either the p200 or the p20 pipette to dispense 20 L, the p20 pipette will

likely be much more accurate at this volume.

PARTS OF THE MICROPIPETTES

Before beginning to use the pipettes, it is

best to familiarize yourself with the basic

parts (shown in the figure on the right).

The operation button is used to aspirate(draw up) liquid into disposable tips that

fit over the ends of the barrels and then

to dispense the liquid. The tip eject button

is to get rid of the used pipette tip. The

calibration knob is used to adjust the

volume of liquid that the pipette is set to

dispense. The volume to which the

pipette is set is viewed in the volume set-

point window. The following page

describes how to read this window. Note:

you can distinguish between the pipettes

in three ways, by the labeling on the

operating button, by the color of the

operating button, and by the size of the

barrel.

Reminder: 1 mL = 1000 L

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Reading the volume set-point window

People that are new to using micropipettes

often find reading the volume set-point

window to be confusing, partly because

the windows look a little different for thevarious pipettes. The volume set-point

windows for the three different size

pipettes are shown to the right. The red

lines on the pipettes have been

accentuated here - note there is no

horizontal red line on the p200 pipette.

p20 pipette

The top two digits tell the number ofmicroliters that the pipette will dispense,

while the number below the red line

indicates the tenths of microliters. The tick

marks between the tenths indicate hundredths of microliters. The example window

indicates that the pipette is set to a volume of 19.50 microliters (you can read two

decimal places with the p20 pipette).

p200 pipette

The three digits on the p200 pipette indicates the number of microliters. The tick marks

between the bottom digits indicate tenths of microliters. The example window indicates

that the pipette is set to a volume of 98.0 microliters (you can read one decimal place

with the p200 pipette).

p1000 pipette

The number above the red line indicates the milliliters that the pipette is set to. This digit

will read either zero or one, as the maximum volume that it can dispense is one milliliter.

The two digits below the red line indicate the hundreds and tens of microliters being

dispensed. The tick marks between the tens indicates the individual microliters being

dispensed. This pipette is set to dispense 650 microliters (you cannot read any digits tothe right of the decimal point with the p1000 pipette).

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METHOD FOR USE

1) Use the information above to select the correct size pipette. Turn the calibration

knob until the display reads the correct volume. NEVER USE A PIPETTE OUTSIDE

THE RANGE FOR WHICH IT IS SPECIFIED TO BE USE.

2) Press the barrel of the pipette down onto the correct size of disposable tip. The p20

and p200 can use the same size tip (although not all tips fit both size pipettes), while

the p1000 always uses a larger tip. Make sure that the tip forms a complete seal

around the barrel of the pipette. NEVER USE A PIPETTE WITHOUT A TIP

ATTACHED as serious damage will result to the pipette and the incorrect volume of

liquid will be dispensed. If you accidentally pipette liquid into the barrel of the pipette,

immediately inform your instructor so that they can clean and/or repair the pipette.

3) Depress the button on the top of the pipette to the first stop. Then, holding the

pipette vertically, immerse the tip into the liquid to a depth of 3-5 mm. It is importantthat you do not submerge more of the tip into the liquid, as liquid will stick to the

outside of the tip, be dispensed into the target vessel, adversely affecting your

pipetting accuracy.

4) Slowly release the button to aspirate the specified volume of fluid into the tip. It is

crucial that you do this fairly slowly (especially with the larger pipettes) as releasing

the button too quickly can cause air bubbles to form and may result in liquid being

pulled into the barrel of the pipette.

5) Pull the pipette straight up to pull the tip from the liquid. If there is any liquid on the

outside of the tip, you may gently touch the tip to the inside of the container from

which you are pipetting in order to remove the excess liquid. Do not wipe the outside

of the tip with a tissue, Kim wipe, or paper towel.

6) Place the tip into the vessel that you wish to dispense the liquid into. If there is

already liquid in the vessel, then immerse the tip into the liquid to a depth of 3-5 mm

and then press the button all the way to the second stop. Remove the tip while

holding the bottom down to the second stop. If there is not already liquid in the

vessel, then gently press the tip against the inside of the bottom of the vessel and

press the button to the second stop and remove the pipette while holding down the

button.

7) Press the eject button to eject the tip into an appropriate container.

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ADDITIONAL OPERATING HINTS

A) Make sure that the tip is in place and correctly fitted. A poor fitting tip will leak and

will result in inaccurate pipetting.

B) Aspirating and dispensing of liquids should be done in a slow, controlled manner.Pipetting too quickly results in inaccuracy and can potentially cause liquids to be

sucked up into the pipette and damage it.

C) Always hold the pipette vertically when in use. Do not hold the pipette horizontally or

set the pipette down on the bench when it is holding liquids, as the liquids can run

into the barrel of the pipette, causing contamination/damage. This is especially

important for the p1000 pipette.

D) If you are pipetting viscous liquids, such as buffers containing glycerol, it may be

helpful to pre-wet the inside of the tip by pipetting up and down once with the liquid

before performing the actual volume measurement.

E) It is often necessary to mix the components as you add them together. The two most

commonly used techniques for mixing while adding components include pipetting up

and down and stirring with the pipette tip. Remember: if you are working with

enzymes or other properly folded proteins, you will want to mix very gently.

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Checking the Accuracy and Precision of Pipettes

Water has a density that varies with temperature, with room temperature water (22C)

having a density of 0.99777 g/mL. You will use this constant to determine the accuracy

of the pipettes that you have been assigned. The protocol that you will use will also giveyou a measure of your pipetting precision. Remember: accuracy is how close the

average volume the pipette dispenses is to an intended volume whereas precision is

how close the volumes that the pipette dispenses are to each other. You may have

seen the following example describing accuracy and precision:

Equipment and Reagents Needed

A clean 50 mL beaker containing 40 mL of distilled water (white tap) A screw top vial (obtained from the instructor)

The p1000, p200, and p20 micropipettes

Disposable tips for the three pipettes

A 100-200 mL beaker that will be used to collect used pipette tips

Analytical balance

Calculator

Microsoft Excel can make the calculations much easier to complete.

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Protocol

Step 1: Collect the equipment and reagents listed and find an analytical balance to

work at.

Step 2: Pour or pipette about a milliliter of water into the screw top vial. Place the vialon the analytical balance and tare (zero) the balance.

Step 3: Check the accuracy of each pipette at two different volumes (according to the

table below) to be sure that it is calibrated and functioning properly. When

dispensing the water into the vial, make sure to place the tip into the water in

the vial and then press the operating button to the second stop. Remove the

tip from the water while holding the button down to the second stop.

Step 4: Enter your data into Microsoft Excel and calculate the mean mass of the

water pipette, the standard deviation (SD) of the mean, the coefficient of

variation (CV), and the accuracy of each pipette for the two volumes used.

The CV should be less than 10%. Consult with your instructor if the CV is

greater than 10%.

CV calculation: SD / mean X 100%

Accuracy calculation:

1) Calculate theoretical mass of water by multiplying the volume (in mL) by

0.99777 g/mL (for example, 0.950 mL X 0.99777 g/mL = 0.948 g)

2) use the following equation to calculate the accuracy:

100% [ABS(mean mass - theoretical mass) / theoretical mass X 100%]

(ABS means take the absolute value of the value in parentheses)

An example Excel spreadsheet is posted to the course blackboard

site. Use it at your own risk. Whenever you use an excel

spreadsheet to calculate your data, double check that the formulasare correct and that your values are being calculated correctly. Also,

double check that you have entered your data correctly.

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Data Collection Sheet

p1000 pipette

volume = 950 L

mass (g) ________ ________ ________ ________ ________

________ ________ ________ ________ ________

Mean = _________ SD = ___________

CV = _________ Accuracy = ___________

volume = 525 L

mass (g) ________ ________ ________ ________ ________

________ ________ ________ ________ ________

Mean = _________ SD = ___________

CV = _________ Accuracy = ___________

p200 pipette

volume = 200 L

mass (g) ________ ________ ________ ________ ________

________ ________ ________ ________ ________

Mean = _________ SD = ___________

CV = _________ Accuracy = ___________

volume = 105 L

mass (g) ________ ________ ________ ________ ________

________ ________ ________ ________ ________

Mean = _________ SD = ___________

CV = _________ Accuracy = ___________

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p20 pipette

volume = 19.5 L

mass (g) ________ ________ ________ ________ ________

________ ________ ________ ________ ________

Mean = _________ SD = ___________

CV = _________ Accuracy = ___________

volume = 7.5 L

mass (g) ________ ________ ________ ________ ________

________ ________ ________ ________ ________

Mean = _________ SD = ___________

CV = _________ Accuracy = ___________

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Quantitative Measurement of Proteins

Spectrophotometry and colorimetry are analytical methods of measuring theamount of light absorbed by a substance in solution. These methods are commonly

used to measure the quantity of biochemical materials. All substances in solutionabsorb light of certain wavelengths and then transmit the light at different wavelengths.

Absorbance is a physical property of a substance just like the melting point or boilingpoint. Absorbance can be used to measure the amount of a given substance in solutionby utilizing the Beer-Lambert law:

A = lC

where

A = absorbance at the given wavelength = molar extinction coefficient

l = path length in cm (almost always 1)C = molar concentration of chromophore

The Beer-Lambert law requires the use of a specified wavelength of light. Generally, thewavelength of light used is the wavelength at which the substance has the greatestabsorbance (the peak) is used and this can be determined by performing a scan over agiven range of wavelengths (shown in Figure 1).

If the extinction coefficient for a substance at the maximum absorbance is knownand the path length is fixed, the concentration of the substance can be determined. The

extinction coefficient may be obtained from the literature or determined by measuringthe absorbance at different concentrations of the substance. A plot of the absorbanceversus concentration should give a linear plot whose slope is the molar extinctioncoefficient (for that unit of protein concentration) when the cell length is 1.00 cm.

Absorption occurs when photon energies in the visible or ultraviolet (UV) regionscause electronic transitions in a molecule. The absorption of visible and ultraviolet lightby organic compounds usually occurs when there is unsaturation in the molecule. A

Figure 1.An absorption spectrum of a given substance over arange of wavelengths from 195 nm to 315 nm. The maximumabsorbance for this substance is approximately 234 nm. If theconcentration of this substance is known, the resultingabsorbance at this wavelength can be used to calculate themolar extinction coefficient (). Scan taken from V.A. McKie, et.al (2001) Biochem. J. 355: 167-177.

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specific group of atoms having unsaturation and absorbing light is called a chromophoreor chromophoric group. Common chromophoric groups include carbon-carbon doubleand triple bonds, carbonyl, carboxyl, amide, azo, nitrile, nitroso, nitro, imidazole, indole,purine, and pyrimidine groups. Any molecule containing one or more such groups hasan absorption band somewhere in the visible or ultraviolet regions. Conjugated doublebonds also contribute to specific absorption bands, causing a decrease in the energyrequired for a transition and producing a peak at higher wavelengths.

Chemical and enzymatic reactions are sometimes designed to generate asubstance that has an absorption maximum in the visible or UV range; such substancescan readily be quantified, providing a convenient way to measure the reaction rate. Asyou will see later this quarter, this may involve the addition of compound that indicatesoxidation/reduction, dehydration/condensation, or the addition of a complexing orchelating agent. The type of reaction is determined by the particular chemistry of thesubstance being measured.

In the following set of exercises, you will use three different protein assay

techniques to quantitate the concentration of proteins in samples of unknownconcentrations. It is important that you understand that these three techniquesquantitate the total amount of protein in the samples and are not able to determine theconcentration of a specific protein from a mixture of polypeptides. Other techniques,with ELISA (enzyme-linked immunosorbant assay) being the most common, are used toquantify the content of a specific protein in a mixture of proteins. The majority of thesemore precise techniques utilize the specific binding of antibodies in order to distinguishbetween the given protein and the other nonspecific proteins present in the sample.

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Bradford Method to Measure Protein Concentration

The laboratory practices of protein purification, enzyme assays, or other biochemicaland cellular analyses require a rapid and sensitive method for the measurement ofprotein concentration. The Bradford Method1for protein quantitation has gained wideacceptance in the biochemical literature. It is based on the binding of a dye to proteinwith a change in dye color upon protein binding. Coomassie Brilliant Blue G-250 (Fig.1.) is red in solution and blue when complexed with a protein. Coomassie brilliant bluebinds to positively charged side groups on amino acid residues in the protein, especiallyto arginine, and to aromatic amino acids. The binding of the dye to protein is a rapidprocess that takes 10 to 15 minutes. The protein-dye complex then remains stable insolution for a sufficient amount of time to complete the assay, approximately 60 min.Due to the high extinction coefficient of the protein-dye complex, it is a sensitive assayallowing measurement of protein at the g / mL level. You can read more about thisprotein assay by consulting theBio-Rad website.Bio-Rad is a company that sells theCoomassie Brilliant Blue G-250 (Figure 2).

Figure 2. Coomassie Bri lliant Blue G-250

Reference1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding.Anal Biochem72:248-54.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_9004.pdfhttp://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_9004.pdfhttp://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_9004.pdfhttp://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_9004.pdf

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A beaker that will be used to as a waste container

1.6 mL microcentrifuge tubes

Microcentrifuge rack Bio-Rad Bradford Reagent

0.01% w/v (0.1 mg/mL) Bovine Serum Albumin (BSA)

Sample of unknown BSA concentration

Approximately 20 mL of distilled H2O (from white taps) in a clean beaker

Vortexer

Timer

Plastic cuvette

UV-Vis spectrophotometer

Microsoft Excel

Protocol

Note: Refer to the Appendix for instructions for use of the UV-Visspectrophotometer.

Step 1: Prepare BSA samples for making a standard curve by obtaining 1.0 mL of the0.01% BSA stock solution and making the following dilutions in 1.6 mLmicrocentrifuge tubes according to the table below. Before you begin

pipetting, label the caps of your microcentrifuge tubes with: 0, 2, 4, 6,8, 10, 12. Pipette 1.0 mL of distilled water and dispense it into the tubelabeled 0. This tube will be used as your blank. For the remaining tubesadd the water first, followed by pipetting the BSA directly into the water(pipette up and down several times to get all of the BSA out of the pipette tip).Mix the components by inverting the tubes 10 times.

H2OmL

Stock BSA volumemL

[BSA] after dilutiong protein /mL

1.000 0.000 0.00

0.980 0.020 2.000.960 0.040 4.00

0.940 0.060 6.000.920 0.080 8.000.900 0.100 10.0

0.880 0.120 12.0

Step 2: Label the top of three fresh microcentrifuge tubes with: U1, U2, and U3.Obtain your unknown sample from the refrigerator (It will be labeled as Br#;with # representing a number). Make sure you write down the number of yourunknown. Without changing the tip, pipette 1.0 mL of your unknown sampleinto the three tubes labeled U1, U2, and U3. You will assay the

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concentration of the same unknown three times (a triplicate determination).

Step 3: Transfer by pouring 3.0 mL of Bradford reagent (it is located in the refrigeratorand has a yellow label that says Bio-Rad Protein Assay Reagent) into 10 mLgraduated cylinder. You may then transfer the 3 mL of Bradford reagent into asmall clean beaker for pipette access. Use the p1000 pipette to add 0.225 mLof the Bradford reagent to all the tubes in Steps 1 and 2, i.e., the water blank,

the known BSA standards, and the unknown BSA samples to bring the totalvolume up to 1.225 mL. Mix by vortexing or inverting 10 times. Important: theBradford method is time-sensitive and the color reaction changes over time.Thus you must work in a timely manner.

Step 4: Incubate the Bradford reagent and protein mixtures for 10 minutes on thebench top (at room temperature). This is a good time to review the aminoacids. Be sure to measure the absorbance of your solutions in an efficientmanner within 15 minutes after the incubation period.

Step 5: Take your samples, a water bottle, a waste container, and a disposable

cuvette to a spectrophotometer. Check that the spectrophotometer is set toread 595 nm. Pour the contents of the tube labeled 0 into the cuvette andplace the cuvette in the spectrophotometer. Make sure that the cuvette ispositioned so that the clear windows are on the left and right side, not to thefront and back.

Step 6: Close the door and zero (blank) the spectrophotometer. The instrumentshould now read 0.000. Once the instrument is zeroed, read the absorbanceat 595 nm of the standard curve protein samples from lowest to highestconcentration, then read your unknowns. Use one cuvette for all of yourmeasurements, rinsing with water in between samples (make sure to give the

cuvette a good shake to remove water from the cuvette or tap it on a set ofpaper towels). Pour the next solution to be measured into the cuvette, place itinto the spectrophotometer, and record the absorbance in your laboratorynotebook. As you work please wipe up any spills with a paper towel.

Step 7: Plot absorbance at 595 nm (y-axis) versus protein concentration in g/mL (x-axis) of the samples of known protein concentration. Use Excel linearregression analysis to determine the best straight line to fit the data points,the equation of the line and the R-squared value. Using the equation of theline and the unknown sample absorbance value, calculate the unknownprotein concentration for each sample, and then the average and standard

deviation and coefficient of variation. Be sure to identify by number theunknown sample you used.

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Ultraviolet Absorption to Measure Protein Concentration

Proteins have an absorption spectrum in the ultraviolet region. Below 230 nm,the absorption of a protein rises rapidly, reaching a maximum around 190 nm due to theabsorption of the peptide bond. Other substances, such as carboxylic acids, buffer ions,and alcohols, also absorb in this region and interfere with the measurement, making itless specific for proteins.

Proteins also have an absorption peak near 275-280 nm due to their aromatictyrosine and tryptophan residues; the molar extinction coefficients of tryptophan andtyrosine at 280 nm are 5502 and 1209, respectively (phenylalanine has a molarextinction coefficient of only 2 at 280 nm). Each protein has its own extinctioncoefficient, which varies with its amount of tyrosine and tryptophan. The absorbancemeasurements at 280 nm for 1% (w/v) solutions of protein vary from 5 to 60, with manyproteins having values around 10. Absorbance measurements at 280 nm are frequentlyused to obtain an elution profile of a chromatographic separation of proteins on acolumn. The method is relatively fast, sensitive, easily automated, and (mostimportantly) nondestructive. A disadvantage of the method is that many othercompounds also absorb in the 280 nm region, especially nucleic acids, which have a

maximum absorbance around 260 nm. Pure proteins typically have a 280 nm / 260 nmabsorbance ratio of about 1.6, whereas pure nucleic acids have a ratio of about 0.5. Aninitial test of the purity of a protein solution may be obtained by measuring andcalculating the 280/260 ratio. You will calculate the 280/260 ratio of a solution of DNAand protein to determine their relative purity.




1.6 mL microcentrifuge tubes Microcentrifuge rack

1% w/v (10 mg/mL) Bovine Serum Albumin (BSA)

0.25% BSA

0.01% DNA solution


Unknown BSA sample (for UV!)

Squirt bottle filled with dH2O

quartz cuvette


Microsoft Excel

Warning: quartz cuvettes are very fragile and expensive (~$300 each).Rinse the cuvette out with mild soapy water (and then dH2O) prior to and

after use. Do not use a paper towel to dry the cuvette.

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Protocol


Part 1:

Step 1: Gather the following reagents and equipment: a squirt bottle containing dH2O,a test tube holding ~3-5 mL of 0.25% BSA, a test tube holding ~3-5 mL of0.01% DNA solution, a quartz cuvette, and a beaker to hold waste material.Take these materials to an available UV-Vis spectrophotometer.

Step 2: Use the squirt bottle to fill the quartz cuvette with ~2 mL of dH2O and placethe cuvette in the chamber of the UV-Vis spectrophotometer. Perform abaseline (blank) scan from 240-320 nm. This will take about 30 seconds.

Step 3: Pour the water into the waste beaker and then pour ~2 mL of the 0.25% BSA

solution into the cuvette. Press the Scanbutton. Note the large absorbanceband or peak at 280 nm.

Step 4: Use the cursor keys to move the vertical line across the screen to the 280 nmand 260 nm positions. Write down the absorbance shown on the screen forboth wavelengths. You will use these values to calculate a 280/260 ratio forprotein.

Step 5: Pour out the BSA solution into the waste beaker and rinse out the cuvettetwice with dH2O. Pour ~2 mL of the 0.01% DNA solution into the cuvette andrepeat Steps 3 and 4. You will use these values to calculate a 280/260 ratio

for DNA.

Step 6: Repeat step 5 with your unknown solution so that you can calculate its280/260 ratio.

Use this data to calculate the ratio o f the absorbance at 280 nm to that at 260 nmfor each solution to determine relative purity.

Part 2:

Step 1: Obtain 2.0 mL of 1.00% (w/v) BSA (10 mg/mL) from the laboratoryrefrigerator. Label 1.6 mL microcentrifuge tubes with the concentrations listedin the following step.

Step 2: Use the 1% BSA solution to prepare 1.5 mL of each of the following dilutions:0.05%, 0.10%, 0.15%, 0.20%, and 0.25% BSA. Use the dilution equation:CiVi=CfVf, where Ci= initial concentration and C f= final concentration and Viand Vfare initial and final volumes, respectively.

Step 3: Take the following items to an available UV-Vis spectrophotometer: yourdiluted BSA samples, your unknown sample, a squirt bottle with dH2O, a

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quartz cuvette, a waste beaker for rinsing the cuvette and your p1000pipette with a tip. Set up the spectrophotometer (See appendix) so that it isreading a single wavelength of 280 nm.

Step 4: Use the water from the squirt bottle to blank the spectrophotometer. Then,pipette your dilutions from the 1.6 mL microcentrifuge tubes into the cuvette(start with the least concentrated). Read and write down the absorbance for

each sample. Finally, rinse the cuvette once or twice with dH2O and pour ~2-3mL of your unknown BSA sample into the quartz cuvette and measure andrecord its absorbance. Pour out the BSA sample and measure and record theabsorbance for the unknown sample two more times. From these threevalues you will calculate the average and standard deviation. Make sure youwrite down the number of your unknown sample.

Step 5: Plot the absorbance at 280 nm versus concentration for the samples ofknown concentration. Use linear regression to determine the best straight linethrough the data points. From the equation for the line, calculate theconcentration (in %) of the unknown solution.

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Biuret Method to Measure Protein Concentration

Polypeptides and proteins with two or more peptide bonds give a characteristicpurple color when treated with dilute, alkaline copper sulfate solution. The color iscaused by the formation of a complex of copper (II) ion with four nitrogen atoms, twofrom each of two peptide chains (Figure 1). The color intensity is proportional to theconcentration of protein. Ammonia or ammonium ions will interfere with thisdetermination by complexing the copper of the Biuret reagent and giving a falsepositive. The Biuret reaction requires relatively large amounts of protein, 1-20 mg.




1.6 mL microcentrifuge tubes

Microcentrifuge rack

1% w/v (10 mg/mL) Bovine Serum Albumin (BSA)

Biuret reagent


Unknown BSA sample (for Biuret!)

Squirt bottle filled with dH2O plastic cuvette


Microsoft Excel

Figure 1. Protein-copper complex.

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Protocol


Step 1: Obtain about 3 mL of 1.00% (10 mg/mL) BSA from the laboratory refrigerator

by pouring into a clean 10 mL graduated cylinder. Label 1.6 mLmicrocentrifuge tubes with the concentrations listed in step 2.

Step 2: Dilute the 1% solution of BSA to prepare 0.600 mL (= 600 L) samplescontaining 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 6.0mg/mL, and 8.0 mg/mL of BSA. Prepare these in the 1.6 mL microcentrifugetubes.

Step 3: Pipette 600 L of dH2O into a microcentrifuge tube for a blank and 600 L ofthe unknown BSA sample to each of the three tubes labeled as unknown.

Step 4: Add 900 L of theBiuret reagent to each of the tubes you prepared in Steps 2and 3, followed by mixing by inversion three times or by vortexing. Incubatethe samples containing Biuret reagent in a 37 C water bath for 10 minutes.Then place the samples in a room temperature water bath for 5 minutes tocool or allow them to cool on the bench top.

Step 5: Take your BSA samples (reacted with Biuret reagent), squirt bottle with dH2O,p1000 pipette, plastic cuvette and a waste beaker to an open UV-Visspectrophotometer. Set the spectrophotometer so that it will read absorbanceat 540 nm.

Step 6: Use the transfer the contents from the microcentrifuge tube labeled blank tothe cuvette, place it in the spectrophotometer and blank the instrument.Transfer the contents of the cuvette back to the tube (or to the waste beaker),then begin reading the absorbance of the standard curve samples. Writedown the absorbance values obtained. Rinse the cuvette out with water andthen read the absorbance of the unknown samples.

Step 7: Plot the absorbance at 540 nm versus concentration for the samples ofknown concentration. Use linear regression to determine the best straight linethrough the data points. From the equation for the line, calculate theconcentration (in %) of the unknown solution.

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Grading Rubric: Quantitative Measurement of Protein (Formal Report)

Introduction (10 points):

State the purpose of this experiment, including a reasonable rationale for why you learned

three different techniques for determining protein concentration (2).

Describe the fundamental chemical principles involved for each of the three protein

concentration assays (2 points each).

Explain the relationship between absorbance and concentration and how this relationship is

used to quantitate an unknown protein sample. Invoke Beers law (2).

Methods (2 points):

Cite the lab manual and note any changes you made.

Results (24 points):Make sure to report the numbers of your three unknowns!

Biuret Assay (7 points)

Table listing test tube contents (protein concentration) and absorbance values (1)

Figure showing absorbance vs. concentration using Excel (1). Your figures should be

labeled with a number (e.g., Fig. 1.) and title just as those found in the peer-reviewed

literature. Give your figure a title that describes the data in the figure or the overall purposeof the figure.

r2 value of 0.98 or higher (1)

correct calculation of unknown protein conc. from standard curve (1)

accuracy of calculated protein concentration (3 points)

error:

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Electrophoresis of Serum Proteins

Using SDS-PAGE

Electrophoresis is the movement of charged molecules through asolid substrate that is mediated by the application of an electricfield. Electrophoresis is an important method of separatingbiological molecules because it is highly sensitive to differences incharge, mass, and shape and can separate very large molecules,including proteins, which are the focus of this exercise. As thecharacteristics of each protein affects how it behaves duringelectrophoresis, a brief description of these qualities will first begiven:

Charge:The net charge on different proteins varies widely and is

due to a combination of the amino acid composition of the proteinand the pH of the environment. Acidic amino acids (aspartic andglutamic acid) have a negative charge at physiological pH, whilebasic amino acids (lysine and arginine) each have a positivecharge. Histidine residues, which a have pKa of 6.0, are neutral atpH values above 7.0, but are positively charged (or partiallypositively charged) at lower pH. However, most proteins have acombination of acidic and basic amino acids, which partiallycounteract each other.

Mass:Just as the net charge varies significantly from protein to

protein, the mass of proteins is also disparate. The smallestproteins contain only about 20 amino acids while the largest knownprotein, Titin, is composed of 38,138 residues 2. Given the averagemolecular weight of amino acids of 110 Da, this give Titin anapproximate mass of 4,195,180 Da (~4.2 MDa)! It is important tounderstand where this value of 110 Da/ residue comes from. If onetakes the average molecular mass of each of the 20 amino acids,the average molecular weight is ~135 Da.However, smaller amino acids such asalanine and glycine tend to be moreabundant in proteins compared to large

residues, like as tryptophan and histidine.

This weighted average results in the actualaverage weight being closer to 128 Da.Additionally; an equivalent of one water (18Da) is eliminated from each amino acidduring peptide bond formation (shown on theright), resulting in the average molecularweight of 110 Da. The loss of water during

Daltons(Da)is a

unit of mass that isequal to 1.0 g/mol.

pKais the pH at

which one-half ofan acidic proton is

dissociated.

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peptide bond formation is shown in the figure on the right, where aleucine residue is being added to the C-terminal end of thetripeptide Ala-Val-Gly.

Shape/size:It is important to note here that the absolute three-

dimensional size of any native protein also depends on how tightlypacked it is. Large molecules, such as proteins, take on distinctiveshapes with widely varying length-to-width ratios. The threedimensional shapes are maintained by hydrophobic interactions,electrostatic interactions and disulfide bonds. Most cytoplasmicproteins are globular and have generally round shapes, whilefibrous proteins are elongated.

Returning to electrophoresis, each of these factors can determinethe rate of migration through a solid substrate. The solid substratesused for electrophoresis include materials such as paper, agarose,

and polyacrylamide. The polymerization of acrylamide and bis-acrylamide creates a gel with pore sizes that can be varied bychanging the ratios of these reagents. These pores retard themigration of proteins through the gel in a manner dependent uponboth size and shape. Separation of proteins by polyacrylamide gelelectrophoresis (PAGE) depends on differences in the frictionalcoefficients of the proteins in an electric field and is a function ofsize, shape, and charge. The rate of movement or velocity (v) of amolecule within an electric field is a function of the net charge (z)and frictional coefficient (f) of the molecule and the field strength (E)of the system, thus giving the crude mathematical relationship:

v= Ez/f

There are many applications for which it is necessary or preferableto separate proteins based solely by mass, rather than acombination of charge, mass and shape. Achieving separation onlyby mass is easily accomplished by the inclusion of two chemicals.The first of these is -mercaptoethanol, a powerful reducing agentthat breaks disulfide bonds. This helps to both separate theindividual components of multiple-subunit proteins and to denature

individual polypeptides. The secondreagent is sodium dodecyl sulfate(SDS), a negatively-chargeddetergent that binds to proteinsevenly along the polypeptide in a ratioof one SDS molecule per two aminoacids. This even decoration ofpolypeptides with SDS results in the

MFQPAPKRCFTI ESLVAKDS PLPASRSEDP I RPAALSYAN

SSPI NPFLNG FHSAAAAAAG RGVYSNPDLV FAEAVSHPPN

PAVPVHPVPP PHALAAHPLP SSHSPHPLFA SQQRDPSTFYPWLI HRYRYL GHRFQGNDTS PESFLLHNAL ARKPKRI RTA

FSPSQLLRLE HAFEKNHYVV GAERKQLAHS LSLTETQVKV

WFQNRRTKFK RQKLEEEGSD SQQKKKGTHH I NRWRI ATKQ

ASPEEI DVTS DD

This 252 amino acid protein has 22 acidic (red) and

32 basic (green) residues (net = +10). If it were

decorated with SDS, it would also have ~126

additional negative charges (net -116).

Secondary

structures include

-helices and -sheets. Tertiary

structures are

determined by how

these secondary

structures fold onto

each other.

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masking of the intrinsic charges from acidic and basic amino acidsand makes the entire polypeptide negatively charged, while thedetergent action of SDS simultaneously denatures the protein bydisrupting hydrogen bonding within the protein. The sum of thesetwo actions of SDS, along with that of -mercaptoethanol, results

in the complete denaturation and linearization of the protein.Electrophoresis performed in the presence of SDS is called SDS-PAGE and resolves proteins according solely to their lengths. Thedistance a particular protein migrates through a gel (Rf) isinversely proportional to the log of the molecular weight (MW):

Rf= 1 / logMW

Once the proteins have been resolved by electrophoresis, theseparated proteins may be visualized in numerous ways, althoughthey are most frequently visualized by staining with chemicals or

specific dyes, as you will do in the following exercise. For differentapplications, gels are not stained but are rather used as apreliminary step in more advanced experimental procedures suchas western blots. It is also possible to purify specific bands ofproteins from polyacrylamide gels. Proteins purified from gels canbe used for polypeptide sequencing, mass spectroscopy (foridentification), or for other purposes.

Each of the compartments of an electrophoresis apparatus containsbuffer and an electrode. The solid support forms a bridge betweenthe two compartments through which charged molecules will movewhen a potential (voltage) is applied. When the size of a particularcomponent of a sample is going to be determined, a separatesample containing a mixture of molecules of known sizes may beresolved by electrophoresis along with the samples; these arefrequently referred to as either markers or ladders. Additionally,charged dye(s) that move through the electric field at a known ratemay be used to monitor the progress of electrophoresis in real time.

The situation at hand

When blood coagulates, the clear material that remains is serum,which contains a mixture of many different types of proteins thatperform a wide range of functions. In this experiment you will useSDS-PAGE to (1) separate the major protein components of humanblood serum, (2) determine the actual concentration of one of theseproteins in the serum, and (3) determine the size of this protein.

Ninety-five percent of serum proteins fall into five main groups andare classified according to their mobility in an electrical field under

-mercaptoethanol

is a reducing agent

that breaksdisulfide bonds.

Dithiothreitol

(DTT)also can beused for this

purpose.

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standard (nondenaturing) conditions. Fromhighest to lowest mobility, they are albumin,alpha-1 (1), alpha-2 (2), beta (), and gamma() globulins (Figure 1). The 1, 2, , and -globulins are each composed of groups of

related proteins; -globulins have an especiallywide range of higher molecular weights. Each ofthese classes of protein, apart from albumin, arecomposed of multiple polypeptide chains ofvarious lengths.

In the following experiment you will use SDS-PAGE to (1) separate the protein components ofhuman blood serum, and (2) determine theconcentration of albumin in the serum bycomparing it to a series of serially diluted samples of bovine serum

albumin (BSA). The serially diluted samples will be used to createa standard curve of protein, to which the human albumin band willbe applied in order to quantitate the amount of albumin. You willalso (3) determine the molecular weight of the albumin band. Thisis the most common use of SDS-PAGE and can provide a goodapproximation for the linear length of the polypeptide.

Figure 1. Schematic of the mobility of

the major classes of serum proteins, when

separated by paper electrophoresis

(nondenaturing conditions). Albumin has

the highest mobility, while gammaglobulins are the least mobile. Adapted

from1.

Serial dilution

means that youdilute a sample,

and then use the

diluted sample to

make an even

more diluted

sample, etc.

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Experimental Procedure

Part 1: Loading, running, and staining the gel

Caution: Take note of the warnings that are listed in the right-hand margin.

Note: Pairs of students will share one gel, and each pair ofstudents will share an electrophoresis apparatus with another

group of two.

Step 1:Prepare the Ready Gel (Bio-Rad 7.5% polyacrylamide inTris-HCl buffer): Remove the gel from the package andplace it on a paper towel with the shorter plate facing up.Use a razor blade to cut the tape along the black lineacross the entire bottom of the gel cassette. Pull the tabdiagonally up toward the comb to remove. The tape mustbe completely removed from the bottom of the gel beforethe gel will make contact with the buffer in the lower buffertank; if a sticky film is still in place, use the razor blade thescrape it off. The tape should be left on the sides of the gelas this will hold the plates and gel together.

Step 2:As demonstrated by your instructor, assemble theelectrophoresis apparatus. Fill the inner reservoir betweenthe two gels with 1X Tris/glycine/SDS buffer and then fillthe outer reservoir until the buffer comes into contact withthe bottom of the gel (the slit that was exposed byremoving the tape). Once the buffer has been added,remove the comb from the gel by positioning your thumbson the ridges on each end of the comb and pushingupward slowly with a smooth, continuous motion. Becareful to not pull the comb too quickly, as the resultingsuction can collapse the wells.

Step 3a:Prepare the serum sample in microcentrifuge tubes bymixing the serum proteins (already diluted 10-fold) as

shown in Table1a.

Table 1a. Sample preparation for the human serum.Sample

Description (dilution )Samplevolume

dH2Ovolume

Loading dyevolume

Total volume(final d ilution)

Human blood serum (1:10) 10 L 0 L 10 L 20 L (1:20)

Human blood serum (1:10) 10 L 10 L 20 L 40 L (1:40)

Wear gloves

when handling

polyacrylamide

gels.Unpolymerized

acrylamide is a

potent

neurotoxin.

Read this step

carefully! If youtake the tape off

the sides of the

gel, it will fall

apart.

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Step 3b: Prepare the BSA standards according to Table 1b. Startby labeling microcentrifuge tubes with the finalconcentrations or amounts. Dispense the appropriatevolumes of water into these tubes, then add the BSA. Mix

by pipetting slowly up and down three times, making surethat you do not blow bubbles out of the pipette tip. Donot vortex. Begin by preparing the most concentratedsample (3.2 g/L) and then use this to prepare the 1.6g/L sample. Continue preparing the serial dilutions untilall have been prepared. Then label a second set of tubeswith the amounts of BSA (16 g down to 1 g). Transferthe appropriate amount of diluted BSA into these tubesand then add 10 L of loading dye to each. Note: thevolume total column in Table 1b refers to the volume inthese samples before you transfer some to the next serial

dilution tube (not the final volume you should have).

Table 1b. Sample preparation for the protein standard curve.

source of BSAvolumedH2O

volumeBSA

volumetotal

[finalBSA]

In a separate set of tubesprep your samples

for loading on the gel

10 mg/ml stock(1.00%) 27.2 L 12.8 L 40 L 3.2 g/L none

3.2 g/L 20 L 20 L 40 L 1.6 g/L 10 L + 10 L of loading dye


0.8 g /L 20 L 20 L 40 L 0.4 g/L 10 L + 10 L of loading dye



Step 4:Check the level of the buffer between the gels: it shouldstill be up to the top of the glass plates. If it has leaked alittle, refill with 1X Tris/glycine/SDS buffer. If the buffer hasleaked a lot, ask your instructor to inspect your apparatus.Load samples into the wells of the gel by positioning thepipette tip loaded with the correct amount of sample andloading dye over the top center of a well. The inner plate(in contact with the inner chamber) of the ready gel isshorter than the outer plate. This allows for a ledge togently rest the pipette on while dispensing the sample intothe well. The sample must be dispensed slowly into thewell in order to allow displacement of the buffer withoutswirling and overflow. Note: the molecular weight (MW)markers already are mixed with loading dye. Also, if youchange the samples loaded into any of the wells, makesure to make a note of the change(s) in your notebook.

Make sure to

wear your

gloves - the

running buffercontains

methanol.

Having a hard

time loading the

lanes? Make

sure you took

the comb out.

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Table 2. Sample volumes to load on the gel.

well # volume of protein sample to load groupmember

1 5 L Human blood serum sample (1:40) 1


3 empty


5 20 L MW standards 1

6 20 L 0.1 g/L sample (1 g BSA) 2

7 20 L 0.2 g/L sample (2 g BSA) 2

8 20 L0.4 g/L sample (4 g BSA) 2

9 20 L 0.8 g/L sample (8 g BSA) 2

10 20 L 1.6 g/L sample (16 g BSA) 2

Step 5: Place the lid onto the apparatus and plug the leads intothe power supply. Run the gel at a constant 200V for ~35minutes. Every 5 minutes, check that the buffer in theinner reservoir has not leaked and that the samples arerunning fairly evenly from lanes 1 to 10. Shut off theelectrophoresis power supply once the blue tracking dyehas migrated off the gel.

Step 6: Stain the proteins in the gel as follows: Pour Coomassieblue-250 staining solution into the plastic storage box (in

your drawer) to a depth of approximately 1.0 cm. Whilewearing gloves, remove the gel from the Ready GelCassette: pull off the tape from the sides of the plates andthen gently use a razor blade to pry apart the two plates.Then, float the gel off of the plate by inverting the gel andplate in the staining solution and agitate gently until the gelseparates from the plate.

Step 7: Place the lid on top of the storage box (leave it loose).Place the box in the microwave and heat the gel for 1minute. Place the box on the orbital shaker and allow the

dye to penetrate the gel for an additional five minutes.During this time, wash the electrophoresis apparatus withwarm tap water and rinse 3 times with DI water. Set theapparatus on paper towels on the lab countertop to dry.

Step 8:After staining for an additional 5 minutes (wearing gloves)carefully press the gel to the bottom of the box whilepouring the staining solution back into the original staining

Make sure to

wear your

gloves - theCoomassie stain

contains

methanol.

The current

used for

electrophoresis

is dangerous.

Take the

appropriate

precautions.

Warning: the

staining solution

will be hot after

it has beenmicrowaved.

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solution bottle (gently place a finger on top of the gel tokeep it from slipping into the bottle).

Step 9:Add approximately ~10 mL of destaining solution (50%

MeOH, 10% Glacial Acetic Acid) to the gel in the box andplace the box on the orbital shaker for 3 minutes. Carefullypour off the destain solution into the waste container, add~20 mL of destain solution to the box and place it on theshaker for 10 minutes. Repeat this destain step. Then,pour off the destaining solution into waste and add 50 mLdeionized water, and place it on the benchtop for 10 min. Ifthe gel is still too dark (has a dark blue background makingit difficult to see the protein bands), pour off the water andadd ~20 mL destain solution again for 10 mins. Thenrepeat with ~50 mL deionized water. Allow at least 30

minutes for the gel to equilibrate with dH2O, before you dryit in cellophane. Set up the gel for drying in cellophane asdescribed next.

Make sure to

wear yourgloves - the

destain solution

contains

methanol.

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Drying polyacrylamide gels

in cellophane

Step 1: If your gel is in destain, exchange the destain solution withdistilled water and allow the gel to equilibrate for 15minutes before proceeding (the gel will expand a little bit).Use water and paper towels to clean your bench area sothat it is free of dust and debris. Dry the bench.

Step 2:Unroll the cellophane onto the cleaned bench area andplace two gel drying frames, approximately 1 inch apartover the cellophane. Cut the cellophane with a razor bladeor scissors, leaving 1-2 inches of excess cellophane above

and below the frames. Put the remaining roll of cellophaneaway. Set the cut piece of cellophane to the side.

Step 3:Use the squirt bottle filled with deionized water tocompletely wet an area of the bench slightly larger thanthe cut piece of cellophane then gently place thecellophane on the wetted area. Gently flatten thecellophane out on the wetted area and push out all of thebubbles from underneath the cellophane.

Step 4:Pour the polyacrylamide gel, along with some of thewater, onto the bottom half of the cellophane. Place aframe over the bottom half of the cellophane and gentlynudge the gel into the center of the frame. Push anybubbles out from beneath the gel. Remove the gel frame.

Step 5:Grab the top of the cellophane and drag it down overthe gel so that the top of the cellophane is even withthe bottom edge. The gel should now be sandwichedbetween two layers of cellophane. Push air bubblesout from between the two layers of the cellophane.

Step 6:Place a piece of the frame above the cellophaneand drag the cellophane-gel sandwich over theframe.

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Step 7: Place the second piece of the frame above the sandwich.

Step 8:Gently lift the frames and gel and use four binder clips tohold the ensemble together. The two binder clips on thebottom of the ensemble should be facing down, so thatthe handles of the clips can be used to hold the gel in avertical position. Place the gel in the fume hood overnightso that it can dry completely.

Step 9:Once the gel is dry, remove the clips and use a pair ofscissors or a razor blade to cut off the excess cellophane.Make sure to leave between and 1 inch of cellophane on

each side of the gel. The gel can be stored indefinitely inthis manner and you can use a flatbed scanner to obtainan image of the gel (as a .tif or .jpg file).

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Analyzing the gel using the

ImageJ software

Part I: Quantitating the concentration of human albuminImage J is free software from the National Insti tutes of Health3.

You may download this software on your personalcomputer to do the analysis at home or you may usethe third floor university computer lab in the sciencebuild ing where the software is already downloaded.

Step 1:Open the image file of the gel in ImageJ 3. Inspect theimage, in particular the lane from the standard curve withthe greatest amount of protein and the human serum lanethat you intend to analyze (the first serum lane was used in

the example, but you may wish to use the lane with lessprotein). Use the rectangular selection tool to draw a boxaround the wider of these lanes. Most likely, it will be thelane containing the serum proteins.

Step 2:Once you have made the box, use the left and right arrowkeys to move the box over the lane containing the leastamount of BSA, and press the 1 key at the top of thekeyboard the number pad does not work for this function(the results of this action are shown in Figure 2).

Figure 2. Selection of the first lane to be used for the protein

quantitation analysis. The lanes are numbered above the

gel and reflects the contents as shown in Table 2. Lanes

6 is used because it has the lowest amount of BSA. The

1 button is used to instruct the program to use this as

the first lane for analysis (bound by a green box).

Although you

can rotate the

image within

ImageJ, it is

easier if the

scanned image is

initially straight.

The rectangle

selection tool:

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Step 3:Use the right arrow key on your keyboard to move the box

over the next lane and press the 2 key. Move the box

over the third lane and again press the 2 key. Continue

this process through each of the BSA standard curve lanes

and three more times for the lanes containing the serum

proteins that you wish to analyze (Figure 3). It is OK if the

boxes overlap, as long as each box contains albumin from

only one lane.

Step 4:Press the 3 key to generate the surface plots for thevarious lanes. The plots for the various lanes will appearseparately, with lane #1 on top and the serum proteins atthe bottom. You can use the scroll wheel on your mouse tomove up and down through the various plots.

Figure 3. Selection of the other lanes for protein quantitation.

Lanes 6-10 (BSA) are selected first, followed by the

lanes with the human serum (1-4). Lane 5 contains MW

markers. For the lanes containing BSA, it is ok if the

boxes overlap, as long as each box contains the protein

for only one lane. The bracketed region (*) contains

additional immunoglobulin components of the human

serum, while the other bracketed region (#) contains

contaminants that we will see in future steps.

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Step 5:Use the line selection tool to draw horizontal lines throughthe bottom of the BSA peaks (Figure 4). For simplicity, youcan ignore the smaller peaks that result from the upper(high molecular weight) bands in the lanes with the greateramounts of BSA (seen on the left of the plots). For the

samples containing human serum, you will also need todraw vertical lines to separate the peaks from the adjacent1 proteins (like you did in the previous exercise).

Step 6:Scroll back up to the top plot and use the magic wand tool

to choose the regions in each peak. When you get to the

plot of the serum proteins, select the albumin peak. A

Results window will automatically be generated.

Figure 4.Use the straight line tool to mark the bottom of the peaks (blue lines). Theregion below the lines is background. Plots A and B represent the BSA plot

for lane 7 (2 g), both before (A) and after (B) adding the line to subtract for

background. Plots C and D show the same process for lane 10 (16 g), along

with the presence of contaminants (*). For the lanes containing human

serum: E (lane 2) and F (lane 4), a vertical line must also be used to separate

the albumin peak from the immunoglobulin (#) peaks.

The line

selection tool:

The magic wand

tool:

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Step 7:Copy the data to an Excel spreadsheet. Change the firstcolumn of data to represent the actual amount of BSAloaded in each lane (Figure 5).

Step 8: Use the standard curve to calculate the concentration ofthe human serum albumin in units of g/dL (dL = 0.1 L), thestandard units used in medicine. Do not forget tocompensate for the fact that the serum was supplied to youalready diluted 1:10 and you diluted it more. See Table 1aor Table 2 for dilution factors.

Part II: Determining the size of the human albumin

Step 1:Open the image file of the gel in ImageJ [File->Open] or

[Crtl+O].Rotate the image so that the lanes are vertical.

Step 2:Click Analyze-> Gels-> Gel Analyzer Options and make

sure the invert peaks box is checked. Then, using the

Rectangle tool, make a rectangle through the center of the

molecular weight marker lane. You do not need to select

the entire lane.

Step 3:Press the 1 key at the top of the keyboard. A 1 shouldnow appear in the box over the molecular weight marker

lane.

Step 4:Use the arrow keys on your keyboard to move the box over

the lane with the smallest quantity of human serum and

Figure 5.Plotting a standard curve of the pixel counts from the serial dilutions

of BSA. Use a spreadsheet (left) to generate the standard curve

(right). The line equation is then used to determine the amount of

protein in the band of albumin in the human serum samples.

The rectangle

tool:

These steps

should seem

very familiar, asthey are almost

identical to the

first steps of

Part I.

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press the 2 key. A 2 should now be present in the box

over the second lane.

Step 5:Press the 3 key. This brings up a plot representing each

lane. The plot for lane containing the molecular weight

standards is shown in Figure 6.

Step 6:Choose the point selection tool, then hold shift and click

the first peak (250 kDa). This will label the peak as 1. The

label may be difficult to see as it is small and yellow,located near the lower right corner of the box. While still

holding the shift key, continue and click all of the peaks,

except the extras (Figure 7). It is best to click all of the

peaks in order as they are labeled sequentially. Next, scroll

down so that you can see the plot of the human serum

and, while holding the shift key, click the peak for the

albumin.

Figure 6. Plot of the molecular weight standards. The portion of the laneselected is shown (yellow). The higher molecular weight markers

are positioned on the left hand side of the plot. The extras includes

unresolved low molecular weight markers and the loading dye. You

will not use this peak for subsequent analysis.

The point

selection tool:

Figure 7.The marked peaks. The top is the molecular weights markers and the

bottom is the human albumin. Note that the extras peak was not

picked.

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Step 8:Once the peaks have been marked, click Analyze ->

Measure (or press M). A results table appears with thecorresponding x and y values (Figure 8). Copy and Paste

these values into Microsoft Excel.

Step 9:You will now use the known values of the molecular weight

ladder to create a standard curve. Calculate the log10 of the

molecular weights and use these values for the standard

curve (Figure 9). Plot the values using a scatter plot. Rightclick a data point and select Add Trendline. Check the

boxes for Display Equation on chart and Display R-

squared value on chart.

Figure 9.How to analyze the molecular weight markers plot data. Column A

is the known molecular weights of the markers. Column B is the

log10of column A. Column C is the data from column X (Figure

8). Use columns B and C to make the plot shown on the right. Do

not plot the data point corresponding to the human albumin. The

line equation and the R2-value are shown at the upper right.

Figure 8.The results table obtained from the plot of the molecular weight

markers. The x values represent how far the proteins ran. The other

data is unimportant for this analysis. Do not be concerned if some

of the columns (e.i. IntDen) do not appear in your results window.

Row number 7 represents the human albumin.

Instructors: the

analysis will be

even more

accurate if only

the bands ofsimilar MW are

used for this

standard curve

(37-75 kDa).

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Step 10: The equation obtained from plotting the molecular weight

standards can then be used to determine molecular weighthuman albumin band. Plug the number for your human

albumin band (X-value; 420 in this case) as y and solve the

equation for x. Take the antilog (10x) of this value to get the

molecular weight of the human albumin. For the example

given, this corresponds to a molecular weight of 72 kDa.

References

1. Harmening, D. M., Clinical Hematology and Fundamentals ofHemostatis. 2nd ed.; F.A. Davis Company: Philadelphia,

1992; p 657.2. Bang, M. L.; Centner, T.; Fornoff, F.; Geach, A. J.; Gotthardt,

M.; McNabb, M.; Witt, C. C.; Labeit, D.; Gregorio, C. C.;Granzier, H.; Labeit, S., The complete gene sequence oftitin, expression of an unusual approximately 700-kDa titinisoform, and its interaction with obscurin identify a novel Z-line to I-band linking system. Circulation research 2001, 89(11), 1065-72.

3. ImageJ, 1.45; National Institute of Health: Bethesda, MD,2012. http://rsbweb.nih.gov/ij/download.html

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Informal Report Grading Rubric:Electrophoresis of Serum Proteins Using SDS-PAGE

Results (12 poin ts):

(2 pts)Attach a printout of your gel. Label the lanes on this printout as

1-10. In the figure description, describe the contents of each lane.(1 point)Show the table (from Excel) of your standard curve of the BSA

and the human albumin band.

(1 pt)Show the plot for your standard curve. Make sure you have theaxes labeled. Show the trend line, the R2value, and the line equation.

(2 pts)Calculate the concentrations (of the original undiluted sample) ofthe albumin in each of the human serum samples you prepared. Showyour calculations.

(2 pts)Use one of the concentrations of human serum albumin from theprevious question to calculate the total amount of albumin in a typicalperson (2.8 liters of serum).

(2 pts)Show your table (from Excel) of your standard curve of the

molecular weight markers and your estimation of the size (in kDa) of thehuman albumin band.

(1 pt)Use your estimated MW to calculate the number of amino acids inthe human serum protein using the average MW of all amino acidswhich is 110 g/mol.

(1 pt) Using your estimated MW, calculate the molarity of the albumin inthe human serum sample.

Discussion (13 points):

(2 pt)What three properties of biological molecules influence themobility of the molecules in polyacrylamide gel electrophoresis (PAGE)?

(2 pt)SDS (in SDS-PAGE) eliminates two of these three factors.What are these two factors and how does SDS eliminate them?

(1 pt)2-mercaptoethanol is an ingredient in the loading dye. Whataffect does it have on proteins?

(2 pts)You calculated the concentrations for each of the three humanserum samples you prepared. Are the values the same for each ofthese samples? If not, provide a scientific explanation.

(2 pts)At the right is a list of the proteins that make up the markers.Did you see all of the proteins in this list? If not, explain why. Hint: goto the Bio-Rad website (or other website) and find the sizes ofproteins that are resolved on your gel (7.5% polyacrylamide).

(2 pts)Look up the number of amino acids in mature human serumalbumin. Compare the number of amino acids you calculated to thereported number of amino acids you looked up. If there is a significant

difference, provide an explanation.(2 pts)You used bovine serum albumin as a standard for human serum

albumin. Do you think this was appropriate? Could you use this as astandard for any protein? Provide a rational for your answer.

MW Markers250 kDa150 kDa100 kDa75 kDa (pink)50 kDa37 kDa25 kDa (pink)20 kDa15 kDa10 kDa

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Restriction Enzyme Analysis of Circular DNA

Plasmid DNAs are small, circular DNA molecules that exist naturally in some species of

bacteria. Plasmids duplicate independently of chromosomal DNA and are generally not

required for normal cellular metabolism. The small size of plasmids allows them to beeasily handled in a laboratory setting. In fact, a large percentage of biochemical and

biological research depends on cloning DNA fragments into plasmids for further study.

In this experiment, you will analyze a plasmid DNA sample by restriction endonuclease

digestions.

Before todays experiment, you need to have some understanding of this special family

of enzymes that are used in recombinant DNA work. These enzymes are called

restriction endonucleases (restriction refers to the fact that these enzymes are used to

digest foreign DNA often viral to restrict the growth of the invaders; endonuclease

refers to the fact that these enzymes cut in the middle of DNA sequence endo andare present in the nucleus). The figure below shows the general mechanism through

which restriction endonucleases work. Basically, they recognize specific sequences that

have the special characteristic of being palindromic, or having the same sequence in

both directions.

How restriction endonucleases work:

These enzymes recognize palindromic

sequences (shown in color; the direction of

the repeat sequence is indicated by the

arrows). The enzyme in this case EcoRI recognizes this sequence and cuts in a

staggered manner to generate two

fragments. These staggered ends are called

sticky ends.

In the following experiment, you will analyze a circular DNA that you have digested with

different combinations of restriction endonucleases. One of these DNA samples will be

digested with the restriction endonuclease HindIII(as outlined in the figure on the next

page). The recognition sequence for this restriction endonuclease is present in three

positions in the plasmid; digestion of this plasmid with HindIII therefore results in the

generation of three DNA fragments. The size of these fragments can be determined by

separating the DNA pieces on an agarose gel (similar to gelatin), which separates the

DNA fragments by size: small fragments migrate through the agarose gel more quickly

than do larger fragments.

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Figure 1. Digestion of plasmid DNA

with a restriction endonuclease:

(Top) a map of plasmid pGBKT7. The

red one (1) indicates the position of

nucleotide 1 (arbitrarily assigned). Thenucleotide positions of the HindIII

restriction sites are listed in black,

while the distance between these sites

is shown in blue. Digestion of the

plasmid with HindIII results in three

cuts and therefore three DNA

fragments (the position of nucleotide 1

is present in the fragment having a

length of 1498 nucleotides).

(Bottom)a picture of an agarose

electrophoresis gel with bands of the

plasmid DNA after being digested with

HindIII. The first lane has a marker

with known nucleotide lengths (in base

pairs, bp) listed on the left, while the

second lane has the digested plasmid.

Note how the smaller fragments

migrate further towards the bottom of

the gel.

In the following experiment, you will digest a DNA plasmid (pGBKT7) using several

different combinations of restriction endonuclease enzymes. You will then estimate the

position(s) of the sites that the unknown restriction enzymes cut. The unknown enzymes

will cut, either once or twice, in the middle of one of the three fragments (between two of

the HindIIIsites), resulting in the production of four (or five, if it cuts twice) bands. You

will then determine where the unknown enzymes cut.

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Procedure

Caution: Wear gloves when handling ethidium bromide or gels containingethidium bromide. This chemical intercalates between DNA bases and fluoresces

in UV light. It is a known mutagen.

Waste: The buffer can be poured down the sink and the agarose gel can be

disposed of in the trash.

Part 1: Digesting plasmid DNA

You will work in groups of three. There are four restriction endonucleases (threedifferent unknowns) in the freezer. Each member of the group will use HindIIIand oneunknown. No two members of the group should have the same unknown number. Eachgroup member will set up three DNA digestions: one with HindIIIalone, one with theunknown enzyme alone, and one using both enzymes simultaneously.

Step 1: Use the information in the next paragraph to finish filling out the following table:

Table 1. Components of the DNA digest reactions.

(--------------------------Volumes-------------------------)

ReactionComponent

Reaction #1 Reaction #2 Reaction #3

pGBKT7 2 L 2 L 2 L

10X buffer 2 L( ) 2 L( ) 2 L( )

0.25% BSA 2 L 2 L 2 L

Enzyme #1 2 L( ) 2 L( ) 2 L( )

Enzyme #2 _ L( ) _ L( ) _ L( )

dH2O __L __L __L

Total volume 20 L 20 L 20 L

Reaction numbers 1 and 2 are known as single digests because only one enzyme isused to cut the DNA. In Reaction #1 use only HindIII(write HindIIIin the parentheses

for enzyme #1), and put a zero for enzyme #2 volume. Calculate the volume of dH2O

needed to make the total volume of Reaction #1 equal to 20 L. In Reaction #2 use

only your unknown enzyme (write the unknown number in the parentheses for

enzyme #1), and put a zero for the volume of enzyme #2. Reaction number 3 is a

double digest because two enzymes are used to cut the DNA. For reaction #3, use

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2 L of HindIIIand 2 L of your unknown. Fill out the table above with these volumes

and enzymes. Use Table 2. below to choose the best buffer for each digest and write

the number of the buffer in the 10X buffer parentheses in Table 1. Use the completed

Table 1. as a guide when adding the each reaction component to the individual

microfuge tubes.

Single digest + HindIIIDouble digest

HindIII 2unknown #1 2 2

unknown #2 4 2

unknown #3 3 2

Step 2: Label three microcentrifuge tubes with your initials and the number of thecorresponding reactions. For instance, Todd Krolls first tube would be labeledTK#1. You may write more details on the side of the tube if you wish. BSA is anenzyme stabilizing agent. Completely thaw and mix the buffer(s) and the BSAthat you need before adding the restriction enzyme. Mix by slowly pipetting theentire volume up and down within the pipette tip. Be careful to NOT blow airout of the pipette tip so that bubbles do not form in your solution.

Always KEEP ENZYMES ON ICE when they are not in the freezer.

Step 3:Begin pipetting! Dispense the appropriate volumes of the reaction components(in the first table) into the appropriate microcentrifuge tubes. Add the dH2O first,and then start at the top of the list and add each component with gentle mixing.Mixing should be accomplished by gently pipetting up and down. Try to get asfew air bubbles in the mixture as possible, as air bubbles denature protein (therestriction endonucleases are protein!).

Step 4:When you have assembled the digestion reactions, place the microcentrifugetubes in a flotation device in a 37 C water bath. Allow the digests to go for atleast 45 minutes, with an hour being preferred.

Part 2: Preparing, Running, and Analyzing the Agarose Gel

Step 1: Prepare an agarose gel. One group of three students will share a singleagarose gel and you should all work together to prepare it. You will prepare a45 mL volume gel that contains 1% agarose in Tris-Acetate-EDTA (TAE) buffer.Recall from general chemistry that percentages are sometimes used to preparesolutions. The calculation for preparing a 40 mL 1 % agarose gel is as follows:

Table 2. Best buffer to use for each reaction.

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Weigh out the appropriate amount

Chem 431 Lab Manual w 2016

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