BIOCHEMISTRY EXPERIMENT SSheng Zhao ( 赵晟 ) WWeb for lecture slides ： EEmail/QQ /MSN/gchat: [email protected] or [email protected].

BIOCHEMISTRY EXPERIMENT

Sheng Zhao (赵晟 )

Web for lecture slides：http://teaching.ewindup.info

Email/QQ /MSN/gchat:[email protected] or [email protected]

Mobile:18551669724 or 13675130010

Principle + Practice

Logic analysis

IntroductionFrom public criticism: some schools teach current students by past knowledge to face the future world.

How to face the future？：deal with the real world.

How to deal with the real world?Data and facts

Professor John Ross: fighting with rumors using the data and facts on Sina Weibo, the Chinese Twitter.

As foreign students: conduct experiments carefully and no cheat on exam.

Principle + practice logic analysis

Introduction

Biochemistry: Biology

Chemistry

Molecular level

Biologic structures and functions.

Major methods: Laboratory assays

Interdisciplinary

Examination for Biochemistry class

1. Class participation: 20%

Lecture and experimental training, logic

analysis, and experiment reports.

2. Experiential exam: 10%

Perform a specified experiment

independently.

3. Exam on biochemistry theory (Lecture): 70%

Preview.

Wear the white coat.

Be on time.

Reagents back to place after usage.

Welcome for any question.

Finish report in class.

Clean the room after class.

Requirement

1.Title;

2.Principle；3.Procedures(simple)；4.Results and analysis;

5.Conclusions.

Report (Weibo/Twitter style)

Using the pipette

Adjustable ：0.5-2ΜL, 2-20ΜL, 20-200ΜL,

100-1000ΜL, 1000- 5000ΜL

Usage：

1.Select the right pipette and tips

2.Holding of pipette

3.Sampling

4.expelling of liquid

5.Discharge the pipette tip

Class syllabus by logic

Protein quantification: Folin phenol (Lowry) assay

Ion exchange chromatography for the mixed amino

acids

Transamination

Protein purification

Gel filtration chromatography

Sodium dodecyl sulfate-Polyacrylamide gel

electrophoresis (SDS-PAGE)

Serum triglyceride (TG) measurement

Plasmid DNA extraction, restriction enzyme

digestion, and agarose gel electrophoresis

Enzyme Km

Serum glutamic-pyruvic transaminase (ALT)

measurement (Exam!)

Protein analysis

Nucleic acid analysis

Protein function

Lipid analysis

Class syllabus by real experiments

1. Introduction and Protein quantification: Folin phenol (Lowry) assay

2. Protein purification: Gel filtration chromatography

3. Plasmid DNA extraction, restriction enzyme digestion, and agarose

gel electrophoresis

4. Enzyme Km

5. Transamination

6. Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-

PAGE)

7. Ion exchange chromatography for the mixed amino acids

8. Serum triglyceride (TG) measurement

9. Serum glutamic-pyruvic transaminase (ALT) measurement (Exam!)

PROTEIN QUANTIFICATION: FOLIN PHENOL (LOWRY) METHOD

Folin phenol (Lowry) assay

Usage of spectrophotometer

Plotting of standard curve

Common methods for protein quantification

1. UV Spectrophotometry

2. Lowry assay

3. Coomassie brilliant blue -G250

Folin phenol (Lowry) assay

Step 1

Under alkaline

condition protein

reacts with Cu and forms

complexDark blue A650

In a certain range the strength of the color is proportional to protein concentration.

Complex reduce the

phenol reagents

Step 2

Spectrophotometry

Lambert―Beer law：

In optics, the Lambert―Beer law relates the absorption of light to

the properties of the material through which the light is travelling.

Advantage

Simple and

sensitive(25-250 μg)

Disadvantage

Time consuming， inte

rfered by a wide variety of

chemicals

Sensitivity: 5 µ g/ml，Measurement range: 25~250 µg/ml

1. Use the standard solution.

2. Measure the absorption at a certain wave

length.

3. Plot the standard curve: absorbance as

ordinate and the protein as horizontal

coordinate.

Standard curve

• Cross the zero point

• A is between 0.05 ～ 1.0

650nm

0.125 0.25 0.375 0.5 0.625 mg

Procedure（ 7 tubes）1 2 3 4 5 6 7

Standard solution

— 0.2 0.4 0.6 0.8 1.0 —

Measured sample

— — — — — — 1.0

H2O 1.0 0.8 0.6 0.4 0.2 — —

A 5 5 5 5 5 5 5

Mixing, 10min at RT

B 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Mixing, 30min, 650nm Absorption

Protein content

(μg)

A650nm

REPORT• Title

• Principle

• Procedure

• Results and analysis tube 1 2 3 4 5 6 7

A

Standard curve