Western Blot

Western BlotWestern Blot OROR

protein protein immunoblotimmunoblotN.GhasemnejadN.Ghasemnejad

M.S studentM.S student Iran, shahid Beheshti Medical university , National Iran, shahid Beheshti Medical university , National

Nutrition and Food Technology Research InstituteNutrition and Food Technology Research Institute

What’s western What’s western blot?blot?

The Western blot is an The Western blot is an analytical analytical technigue technigue used to detect used to detect specific specific proteinsproteins in a given in a given sample of tissue homogenate sample of tissue homogenate or extract.or extract.

The name The name Western blotWestern blot was given to the was given to the technique by W. Neal Burnette and is a technique by W. Neal Burnette and is a play on the name southern blot.play on the name southern blot.

Types of blottingTypes of blotting

Southern blotSouthern blot for for DNADNA developed by developed by Edwin SouthernEdwin Southern in 1997. in 1997.

Northern blotNorthern blot for for RNARNA named opposite named opposite of Southern by of Southern by James Alwine and James Alwine and George Stark George Stark

Western blotWestern blot for for ProteinsProteins. Developed . Developed by by George StarkGeorge Stark uses antibodies uses antibodies to locate Proteins. to locate Proteins.

Contents:Contents:Steps in a Western blotSteps in a Western blot

--Tissue preparationTissue preparation --Gel electrophoresisGel electrophoresis -Transfer-Transfer -Blocking -Blocking

-Detection -Detection - - Two stepTwo step - One step- One step

-Analysis -Analysis - Colorimetric detection- Colorimetric detection -- Chemiluminescent Chemiluminescent

detectiondetection - Radioactive detection- Radioactive detection - Fluorescent detection- Fluorescent detection

Tissue preparation:Tissue preparation:-- Samples may be taken from Samples may be taken from whole tissuewhole tissue, from , from cell cell

cultureculture, , bacteriabacteria, , virusvirus or or environmental samplesenvironmental samples..

-- In most cases, samples are solid tissuesIn most cases, samples are solid tissues..

-- firstfirst broken downbroken down mechanicallymechanically using a using a blenderblender (for (for larger sample volumes),larger sample volumes), using a using a homogenizerhomogenizer (smaller volumes),(smaller volumes), or by or by sonication.sonication.

-- Cells may also be Cells may also be broken openbroken open by one of the above by one of the above mechanical methods.mechanical methods.

- - A combination of A combination of biochemicalbiochemical and and mechanicalmechanical techniques, including various types of techniques, including various types of filtrationfiltration and and centrifugationcentrifugation..

- to encourage lysis of cells and to solubilize - to encourage lysis of cells and to solubilize proteins, may be employed :proteins, may be employed :

detergents, salts, and buffersdetergents, salts, and buffers

- - to prevent the digestion of the sample by its own to prevent the digestion of the sample by its own enzymes :enzymes :

Anti Anti Protease and phosphataseProtease and phosphatase

- - to avoid protein denaturing.to avoid protein denaturing.

Tissue preparation is often done at cold Tissue preparation is often done at cold temperaturestemperatures

Gel electrophoresis:Gel electrophoresis:- The proteins of the sample are - The proteins of the sample are separatedseparated using using gel gel

electrophoresis.electrophoresis.

- Separation of proteins may be by - Separation of proteins may be by isoelectric point (pI)isoelectric point (pI) molecular weightmolecular weight, , electric chargeelectric charge, or a , or a combination of combination of these factors.these factors.

- most common type of gel electrophoresis - most common type of gel electrophoresis SDS-PAGE (SDS polyacrylamide gel SDS-PAGE (SDS polyacrylamide gel

electrophoresis) electrophoresis)

- maintains polypeptides in a - maintains polypeptides in a denatured statedenatured state, thus , thus allowsallows

separation of proteins by their separation of proteins by their molecular weightmolecular weight. .

**The The concentration of concentration of acrylamideacrylamide determines the determines the resolution of the gel.resolution of the gel.

- the - the greatergreater the the acrylamide concentrationacrylamide concentration the better the the better the resolution of resolution of lower molecular weight proteinslower molecular weight proteins. .

-The -The lowerlower the the acrylamide concentrationacrylamide concentration the better the the better the resolution of resolution of higher molecular weight proteinshigher molecular weight proteins. .

** Samples are loaded into Samples are loaded into wellswells in the gel.in the gel.

** One lane is usually reserved for a One lane is usually reserved for a markermarker or or ladderladder, , (mixture of proteins having defined molecular weights, (mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands).typically stained so as to form visible, coloured bands).

** When voltage is applied along the gel, proteins migrate When voltage is applied along the gel, proteins migrate into it at different speeds. into it at different speeds. (different (different electrophoretic electrophoretic mobilities).mobilities).

** Two dimentional gel.Two dimentional gel.

What is Gel What is Gel Electrophoresis?Electrophoresis? Gel ElectrophoresisGel Electrophoresis is is

a group of techniques a group of techniques used to used to separate separate moleculesmolecules based on based on physical physical characteristics such characteristics such as as sizesize, , shapeshape, or , or electric charge. electric charge.

Two types Of Gel ElectrophoresisTwo types Of Gel Electrophoresis

•Agarose ElectrophoresisAgarose Electrophoresis for for DNADNA and and RNARNA

•SDS-PAGE ElectrophoresisSDS-PAGE Electrophoresis For For ProtiensProtiens. .

Polymerized gel:Polymerized gel:-Resolving gels made in 6%, -Resolving gels made in 6%,

10%, 12%, 18%.10%, 12%, 18%.-Stacking Gel up to 5% was -Stacking Gel up to 5% was

added to gel and then the added to gel and then the wells are created.wells are created.

The The percentagepercentage chosen chosen depends on the size of depends on the size of the proteinthe protein that one wishes that one wishes to identify or probe in the to identify or probe in the sample. sample.

The smaller it is the bigger The smaller it is the bigger the percentagethe percentage

Usually boughtUsually bought

Polyacrylamide Gel

Loading the SDS-PAGE Loading the SDS-PAGE gelgel

SDS-PAGESDS-PAGE-SDS linearizes all proteins and gives net -SDS linearizes all proteins and gives net

negative charge.negative charge.

SDS PAGESDS PAGE -The Pink Strands are the -The Pink Strands are the Denatured ProteinsDenatured Proteins. . See See

varying sizesvarying sizes

-They Are traveling to the -They Are traveling to the ++ since they have since they have –– charge due to SDS.charge due to SDS.

-These strands go throught the tunnel and are -These strands go throught the tunnel and are seperated by their seperated by their size.size.

Proteins here are actually linearized.

TransferTransfer- In order to make the proteins - In order to make the proteins accessible to antibody accessible to antibody

detectiondetection::

they are moved from within the gel onto a they are moved from within the gel onto a membrane made of membrane made of nitrocellulosenitrocellulose or or ppolyolyvvinileden inileden ddiiffluorideluoride (PVDF(PVDF)) . The . The membranemembrane is placed on is placed on top of the geltop of the gel, and a , and a stack of filter stack of filter paperspapers placed on placed on top of thattop of that..

- The entire stack is placed in a buffer solution which moves up the - The entire stack is placed in a buffer solution which moves up the

paper by paper by capillary actioncapillary action, bringing the , bringing the proteins with itproteins with it..

- Another method for transferring the proteins is called - Another method for transferring the proteins is called

electrobloting and uses an electric electrobloting and uses an electric currentcurrent to pull proteins from the gel into membrane. to pull proteins from the gel into membrane.

-Both varieties of membrane are chosen for -Both varieties of membrane are chosen for their their non-specific protein binding non-specific protein binding propertiesproperties (i.e. binds all proteins equally (i.e. binds all proteins equally well). Protein binding is based upon well). Protein binding is based upon hydrophobic interactionshydrophobic interactions, , as well as as well as charged interactionscharged interactions between the between the membrane and protein.membrane and protein.

- Nitrocellulose- Nitrocellulose membranes are membranes are cheapercheaper than than PVDFPVDF, but are , but are far far more fragilemore fragile and and do not stand up do not stand up well to repeated probingswell to repeated probings..

One majorOne major difference between difference between nitrocellulosenitrocellulose and and PVDF membranesPVDF membranes relates to the relates to the ability ability of each to support "stripping" antibodies of each to support "stripping" antibodies offoff and and reusing the membrane for reusing the membrane for subsequent antibody probes.subsequent antibody probes.

PVDF is sturdier, and can be more reused.PVDF is sturdier, and can be more reused.

Another difference is that, Another difference is that, unlike unlike nitrocellulosenitrocellulose, , PVDFPVDF must must be soaked inbe soaked in 95% ethanol95% ethanol, , isopropanolisopropanol or or methanol methanol before use. before use.

PVDF membranes also tend to be PVDF membranes also tend to be thickerthicker and and more resistant to damage during usemore resistant to damage during use..

The The uniformityuniformity and overall and overall effectivenesseffectiveness of of transfer of protein transfer of protein from the gel to the membranefrom the gel to the membrane can can be checked by staining the membrane be checked by staining the membrane with with coomassiecoomassie or or ponceau Sponceau S dyes. dyes.

Ponceau SPonceau S is the is the more commonmore common of the of the two, due to Ponceau S's two, due to Ponceau S's higher higher sensitivitysensitivity and its and its water solubilitywater solubility makes makes it easier to it easier to subsequently destain and subsequently destain and probe the membraneprobe the membrane..

Blotting Blotting Blotting used Blotting used

to transfer to transfer the samples the samples from the gel from the gel on to a on to a membrane membrane such as a such as a nylon nylon membranemembrane or or nitrocellulonitrocellulose se membranemembrane..

AnalyzeAnalyzed d througthrough h probinprobing with g with nucleic nucleic acid acid probesprobes or or antiboantibody dy probesprobes..

BlockingBlockingsteps must be taken to steps must be taken to prevent interactions prevent interactions

between the between the membranemembrane and the and the antibodyantibody used used for for detection of the target proteindetection of the target protein (since the (since the antibody is a protein itself)antibody is a protein itself)..

Blocking of non-specific binding is achieved Blocking of non-specific binding is achieved by by placing the membrane in a dilute placing the membrane in a dilute solution of proteinsolution of protein

typically typically BBovin ovin SSerum erum AAlbumin lbumin (BSA)(BSA) or or non-fat dry non-fat dry milkmilk (both are inexpensive)(both are inexpensive), with a , with a minuteminute percentage ofpercentage of detergentdetergent such as such as Tween20Tween20..

** The protein in The protein in the dilute the dilute solution solution attaches to attaches to the membrane the membrane in all placesin all places where the where the target proteins target proteins have not have not attached.attached.

** This reduces This reduces "noise" in the "noise" in the final product final product of the Western of the Western blot, leading blot, leading to to clearer clearer resultsresults, and , and eliminates eliminates false false positivespositives..

DetectionDetectionthe membrane is "probed" for the protein of interest with a the membrane is "probed" for the protein of interest with a

modified antibody modified antibody which is linked to a reporter enzymewhich is linked to a reporter enzyme, , which when exposed to an appropriate substrate drives a which when exposed to an appropriate substrate drives a colourimetric reactioncolourimetric reaction and produces a and produces a colourcolour..

- Two step- Two stepPrimary antibodyPrimary antibody {Antibodies are generated when a host {Antibodies are generated when a host

species or immune cell culture is exposed to the species or immune cell culture is exposed to the protein of interest (or a part thereof) }.protein of interest (or a part thereof) }.

A dilute solution of primary antibody A dilute solution of primary antibody (generally between 0.5 and 5 (generally between 0.5 and 5 micrograms/mL)micrograms/mL) is is incubated with the membraneincubated with the membrane under gentle under gentle agitation for anywhere agitation for anywhere from 30 minutes to overnightfrom 30 minutes to overnight at at different different temperaturestemperatures..

The solution is composed of The solution is composed of buffered saline solutionbuffered saline solution with a with a small percentage of small percentage of detergentdetergent, and sometimes with , and sometimes with powdered milk or BSA.powdered milk or BSA.

Secondary antibodySecondary antibody {After {After rinsing the membranerinsing the membrane to to remove unbound primary antibodyremove unbound primary antibody, the membrane , the membrane is exposed to another antibody, directed at a species-is exposed to another antibody, directed at a species-specific portion of the primary antibody }.specific portion of the primary antibody }.

The secondary antibody The secondary antibody is usually linked to is usually linked to biotinbiotin or or to a to a reporter enzymsreporter enzyms such as such as alkalin alkalin phosphatasephosphatase or or horseradish peroxidasehorseradish peroxidase. This . This means thatmeans that severalseveral secondary antibodies will bind to secondary antibodies will bind to one primary antibody and one primary antibody and enhance the signalenhance the signal..

Most commonly, aMost commonly, a horseradish peroxidase-linked horseradish peroxidase-linked secondarysecondary is used to is used to cleave a chemiluminescent cleave a chemiluminescent agentagent, and the reaction product , and the reaction product produces produces luminescenceluminescence in proportion to the in proportion to the amount of amount of proteinprotein..

A A cheapercheaper but but less sensitiveless sensitive approach utilizes a approach utilizes a 4-4-chloronaphthol stainchloronaphthol stain with with 1% horseradish 1% horseradish peroxidaseperoxidase; ; reaction of peroxide radicals with reaction of peroxide radicals with 4-chloronaphthol4-chloronaphthol produces produces a dark brown staina dark brown stain that can be photographed that can be photographed without using without using specialized photographic filmspecialized photographic film..

- One step- One stepHistorically, the probing process was performed in Historically, the probing process was performed in

two steps because of the two steps because of the relative ease of relative ease of producing primary and secondary antibodiesproducing primary and secondary antibodies in separate processes.in separate processes.

one-step probing systems that would one-step probing systems that would allow the process to occur allow the process to occur fasterfaster and and with with less consumablesless consumables..

This requires a probe antibody which This requires a probe antibody which both both recognizes the protein of interestrecognizes the protein of interest and and contains a detectable labelcontains a detectable label, probes which are , probes which are often available for known often available for known proteins tags.proteins tags.

Analysis :Analysis :In practical terms, not all Westerns reveal In practical terms, not all Westerns reveal

protein only at protein only at one bandone band in a membrane. in a membrane. Size approximations are taken by Size approximations are taken by

comparing the stained bandscomparing the stained bands to that of to that of the the marker or ladder loadedmarker or ladder loaded during during electrophoresiselectrophoresis..

The process is repeated for a The process is repeated for a structural structural

proteinprotein, such as actin or tubulin, that , such as actin or tubulin, that should not change between samples.should not change between samples.

This practice This practice ensures correction for the ensures correction for the amount of total proteinamount of total protein on the membrane in on the membrane in case of case of errorserrors or or incomplete transfersincomplete transfers..

- Colorimetric detection- Colorimetric detection ::

This method depends on This method depends on incubation of the incubation of the Western blot with a substrateWestern blot with a substrate that reacts with that reacts with the reporter enzyme (such as peroxidase)the reporter enzyme (such as peroxidase) that that is bound to the secondary antibody.is bound to the secondary antibody.

This converts the This converts the solublesoluble dye into an dye into an insolubleinsoluble form of a form of a different colordifferent color that precipitates next that precipitates next to the enzyme and thereby stains the to the enzyme and thereby stains the membrane.membrane.

Protein levels are evaluated through Protein levels are evaluated through densitometrydensitometry or or spectrophotometryspectrophotometry..

- Chemiluminescent detection :- Chemiluminescent detection :

This methods depend on This methods depend on incubation of the incubation of the Western blot with a substrateWestern blot with a substrate that will that will luminesce when exposed to the reporter on luminesce when exposed to the reporter on the secondary antibody.the secondary antibody.

The light is then detected by The light is then detected by photographic filmphotographic film, , and more recently by and more recently by CCD camerasCCD cameras. .

The image is analysed by densitometry.The image is analysed by densitometry.

Newer software allows further data analysis Newer software allows further data analysis such as such as molecular weight analysismolecular weight analysis if if appropriate standards are used.appropriate standards are used.

Detection by Chemiluminescence

- Radioactive detection :- Radioactive detection :

Radioactive labels Radioactive labels do not require enzyme do not require enzyme substratessubstrates, but rather allow the , but rather allow the placement of placement of medical X-ray film directly medical X-ray film directly against the western blotagainst the western blot which develops which develops as it is exposed to the label and creates as it is exposed to the label and creates dark regionsdark regions which which correspond to the correspond to the protein bands of interestprotein bands of interest

very expensive, health and safety very expensive, health and safety risks are highrisks are high

- Fluorescent detection :- Fluorescent detection :

The fluorescently labeled probe is The fluorescently labeled probe is excited by excited by lightlight and and the emission of the emission of the excitation is then detected by a the excitation is then detected by a photosensor such as CCD cameraphotosensor such as CCD camera. .

Allows further data analysis such as Allows further data analysis such as molecular weightmolecular weight analysis and a analysis and a quantitative western blot analysisquantitative western blot analysis. .

the the most sensitivemost sensitive detection detection methods for blotting analysis.methods for blotting analysis.

Medical diagnostic Medical diagnostic applications:applications:-The confirmatory HIV test-The confirmatory HIV test

-A Western blot is also used as the -A Western blot is also used as the definitive test for Bovine Spongiform definitive test for Bovine Spongiform Encephalopathy (BSE).Encephalopathy (BSE).

-Some forms of Lyme disease testing -Some forms of Lyme disease testing employ Western blotting.employ Western blotting.

-Western blot can also be used as a -Western blot can also be used as a

confirmatory test for Hepatitis B confirmatory test for Hepatitis B infection.infection.

Creating Western Blot Creating Western Blot StripsStrips

1-HIV lysate proteins are separated by size using gel electrophoresis

2-Proteins are transferred (blotted) onto the surface of a membrane

4-Strips are incubated with patient serum and antihuman IgG conjugated with an enzyme (and chromagen)

3-The membrane iscut into strips

HIV Western Blot Banding HIV Western Blot Banding PatternPattern

envgp160

gp120gp

41

gag p55p18p24

pol p65p51p31

When should WB be used?When should WB be used?

Western Blot assay should not be used as a Western Blot assay should not be used as a screening testscreening test..

WB should be viewed as a WB should be viewed as a supplemental testsupplemental test which can be used to which can be used to confirm positive results confirm positive results obtained from EIA.obtained from EIA.

HOWEVER:HOWEVER:

SpecificitySpecificity is is less thanless than that of EIA that of EIA

A significant number of indeterminate blots A significant number of indeterminate blots are seen in low risk populationsare seen in low risk populations

AdvantagAdvantageses

Specific interaction of antibody and Specific interaction of antibody and antigen can be directly visualized.antigen can be directly visualized.

Disadvantages Technically demanding Expensive Subject to interpretation

Presence or absence of bandsIntensity of those bands

Western Blot

WESTERN BLOT

The article’s titleThe article’s titleExpression of OB mRNA & it’s Expression of OB mRNA & it’s

encoded protein in rodents impact encoded protein in rodents impact of nutrition & obesity.of nutrition & obesity.

Thanks forThanks for

your attentionyour attention