Presented by….

RAKESH S.PARMAR

ROLL NO. :CB-0613

BATCH NO.:2013-2014

M.Sc CANCER BIOLOGY

05/09/2013

Blotting

Blots are techniques for transferring DNA , RNA and

proteins onto a carrier so that they can be

separated, and it often follows the use of a gel-

electrophoresis

Blotting techniques

Southern Blot

It is used to detect DNA.

Northern Blot

It is used to detect RNA.

Western Blot

It is used to detect PROTEIN.

Western blotting is an immunoblotting(protein

detection) technique combining the separation

power of SDS PAGE to assess the presence, amount

and molecular- weight of proteins in cellular or

tissue extracts by using antibodies.

The method is characterised by transfering the protein,which was run on a gel by electrophoresis, onto anitrocellulose membrane.

This approach makes the protein stable on themembrane so that several methods including methods ofdetection and quantity the protein content can beemployed.

In Western Blot proteins are separated by SDS-PAGE

Comb, wellformer (removed)

Back plate

Spacer

Front plate

Separating gel (pH8.8)Stacking gel (pH 6.8)

Sample well

Slab gel design

Anode

Upper bufferCompartments

Gel cassette

Lower bufferCompartments

Cathode

Setting up a vertical gel

The SDS-PAGE is sodium dodesylsulphatepolyacrylamide gel electrophoresis.

SDS-PAGE is widely used in biochemistry, forensics, genetics and molecular biology to separate the proteins according to their electrophoretic mobility.

Discontinuous polyacrylamide gel is commonly used as supporting medium in separating proteins by electrophoresis and sds (lauryl sulphate) to denature the proteins.

Principle of SDS-PAGE

Proteins move in the electric field.

Their relative speed depends on

the charge, size, and shape of the

protein

SDS is a strong anionic detergent,means when dissolved its molecules

have net negative charge within a wide pH range

Polyacrylamide gels are composed of chains of polymerised acrylamide that

are cross linked by agent like

N,N’-methylene-bis-acrylamide

Acrylamide and bisacrylamide used to cast the gel.

Cross links formed by bisacrylamide provides rigidity and tensile strength to gel and forms pores through which sds-polypeptide complex passes.

As bisacrylamide increase,the pore size decreases and pore size increase with decrease of bisacrylamide.

The polymerization reaction is catalyzed by TEMED(Tetramethyl Ethylenediamine ) and

APS (ammonium persulfate) generates free radicals which are necessary for the polymerization reaction

polymerization Cross Link

Link

PAGE

Small molecules move faster than big molecules through the

polyacrylamide gel.

Bigger molecules stays near the well.

ACRYLAMIDE CONCENTRATION (%)

LINEAR RANGE OF SEPARATION (KD)

15 10-43

12 12-60

10 20-80

7.5 36-94

5 57-212

Uses of SDS-PAGE

Two main purposes for examining protein preparations using SDS-PAGE

To determine the molecular weight of a protein and

To observe qualitative and quantitative differences among the proteins from different cell preparations

Other uses include- Identify protein - Identify existence of disulfide bonds- Determine sample purity- Quantify amounts of protein

SDS-PAGE includes two type of gel system:

(1)Running gel &

(2)Stacking gel

According to the system, preparation requires casting two different layers of acrylamide between glass plates.

The lower layer (separating or resolving gel) is responsible for actually separating polypeptides by size.

The upper layer (stacking gel) includes the sample wells.It is designed to sweep up proteins in a sample between

two moving boundaries so that they are compressed (stacked) into µm thin layers when they reach the separating gel.

The samples & the stacking gel contains Tris-cl (pH6.8)

Upper & lower buffer reserviors contain Tris-glycine (pH 8.3)

Resolving gel contains Tris-cl (pH8.8)

The leading edge of the moving boundary formed by chlorideions in the sample and stacking gel and the trailing edge iscomposed of glycine molecules.

Between leading and trailing edge of moving boundary there is azone (gap) of lower conductivity and steeper voltage gradientswhich sweeps the polypeptides from the sample and depositsthem on the surface of the resolving gel.

COMPONENTS/GEL VOLUME 5ML 10ML 15ML 20ML 25ML 30ML

6% GEL

H2O 2.6 5.3 7.9 10.6 13.2 15.9

30% ACRYLAMIDE MIX 1 2 3 4 5 6

1.5 M TRIS (Ph 8.8) 1.3 2.5 3.8 5 6.3 7.5

10% SDS 0.05 0.1 0.15 0.2 0.25 0.3

10% AMMONIUM PERSULPHATE 0.05 0.1 0.15 0.2 0.25 0.3

TEMED 0.004 0.008 0.012 0.016 0.02 0.024

8% GEL

H2O 2.3 4.6 6.9 9.3 11.5 13.9

30% ACRYLAMIDE MIX 1.3 2.7 4 5.3 6.7 8

1.5 M TRIS (Ph 8.8) 1.3 2.5 3.8 5 6.3 7.5

10% SDS 0.05 0.1 0.15 0.2 0.25 0.3

10% AMMONIUM PERSULPHATE 0.05 0.1 0.15 0.2 0.25 0.3

TEMED 0.003 0.006 0.009 0.012 0.015 0.018

10% GEL

H2O 1.9 4 5.9 7.9 9.9 11.9

30% ACRYLAMIDE MIX 1.7 3.3 5 6.7 8.3 10

1.5 M TRIS (Ph 8.8) 1.3 2.5 3.8 5 6.3 7.5

10% SDS 0.05 0.1 0.15 0.2 0.25 0.3

10% AMMONIUM PERSULPHATE 0.05 0.1 0.15 0.2 0.25 0.3

TEMED 0.002 0.004 0.006 0.008 0.01 0.012

Stacking gel

To obtain optimal resolution of proteins, a stacking gel ispoured over the top of the resolving gel.

The stacking gel

-lower concentration of acrylamide (larger pore size),

- lower pH

- different ionic content

This allows the proteins in a lane to be concentrated into atight band before entering the running or resolving gel

produces a gel with tighter or better separated protein bands

components/gel volume 1ml 2ml 3ml 4ml 5ml

H2O 0.68 1.4 2.1 2.7 3.4

30% acrylamide mix 0.17 0.33 0.5 0.67 0.83

1.0M Tris(pH6.8) 0.13 0.25 0.38 0.5 0.63

10% SDS 0.01 0.02 0.03 0.04 0.05

10% ammonium per sulphate 0.01 0.02 0.03 0.04 0.05

TEMED 0.001 0.002 0.003 0.004 0.005

Add 1/100 of fresh 10% ammonium persulfate solution. Swirl gently to mix.

Add 1/1000 of TEMED. Swirl gently to mix.

Pour the solution into the gel cassette. Fill the cassette to a level which will allow the comb to be inserted with 5 mm between the bottom of the wells and the top of the resolving gel.

Overlay the gel with 1-2 mm of isobutanol to exclude oxygen and ensure a flat interface between the resolving and stacking gels.

Allow the gel to polymerize for 30 min.

Pouring of Resolving gel

Pour the stacking gel solution directly onto the surface of the polymerised resolving gel.

Immediately insert a clean Teflon comb into the stacking gel solution ,being carefully to avoid trapping air bubbles.

Add more stacking gel solution to fill the spaces of the comb completely.

Place the gel in a vertical position at room temperature.

1X SDS sample loading buffer

• 50 mM Tris-Cl (pH 6.8)• 100mM dithiothreitol (DTT) /β- mercaptoethanol• 10%(v/v) glycerol • 2% (w/v)SDS • Bromophenol blue

SDS and β-mercaptoethanol denature the protein.

SDS binds with denatured proteins and covers withnegative charge

β-mercaptoethanol is a strong reducing agent. It

eliminates disulfide bonds in proteins by reducing them

(adding hydrogen atoms) and breaks apart tertiary &quanternary structure further denaturing proteins

Dithiothreitol (DTT) is an antioxidant that breaksdisulphide bonds

Tris buffer maintains the pH

Glycerol or sucrose for sample density which makes settle down the sample through electrophoresis buffer to bottom when injected to loading well.

Ionisable tracking dye –bromophenol blue for monitoring electrophoretic run.

While the stacking gel is polymerizing, preparethe samples in the appropriate volume of 1%

SDS gel-loading buffer and heat them to 100°cfor 3 minutes to denature the proteins.

[Note: make sure to run molecular weight markerto know the molecular weights of unknownbands]

Loading the sample after removing teflon comb

Sample loading and Gel run

After loading samples, add electrode-buffer to upper reservoir and lower reservoir.

Electrode buffer provides the ions to conduct the current through the gel.

SDS makes proteins negatively charged that attaches the proteins to the anode.

Therefore in electrophoresis, the current must run from cathode (negatively charged, black) to the anode (positive charged, red).

SDS-PAGE

Assemble sandwitch after the gel has run

Remove gel from the glass plate holders

Place the gel in towbins buffer.

Prepare stack of whatman filter paper on blotting unit

On filter paper place nitrocellulose membrane and on top of it place the gel.

Two type of bloting technique-submerged in transfer buffer (Wet Blotting)

Soaked in transfer buffer (Semi dry method)

Transfer buffer contains Tris, glycine and methanol but no ions because ions will coat the electrodes and destroy the transfer apparatus.

Gel:nitrocellulose must have no air bubbles between them (proteins will escape into the bubble not into the nitrocellulose.)

Second step

Transfer step

The transfer of the proteins onto the nitrocellulose membrane.

The proteins separated on the SDS-PAGE gel are trasferred to the membrane by using electrophoresis. The localization of the proteins do not change.

Transfer apparatusTwo major types

Semi-dry apparatus

No transfer buffer chamber

Only has plates (runs for hours)

Submerge transfer apparatus

Contains a chamber filled with transfer buffer

Electrodes

-Plate electrodes (runs for hours)

-Wire electrodes (runs overnight)

Transfer the protein from the gel to the membrane

Transfer of the proteins fractionated by

SDS-PAGE to a solid support membrane

(Western blotting) can be accomplished

by electroblotting

Running a transfer

Similar to running a gel Negatively charged proteins run towards the anode end of the

transfer apparatus

Always have nitrocellulose on the anode side to capture proteins.

We can also use another membrane like PVDF membrane

Why use it?

-Stronger than nitrocellulose and able to strip phosphate groups off proteins.

-Must soak in methanol first since it doesn’t become wet in water

Transfer

In this procedure, a sandwich of geland solid support membrane(Nitrocellulose or PVDF) iscompressed in a cassette andimmersed in buffer between twoparallel electrodes.

A current is passed at right angles tothe gel, which causes the separatedproteins to electrophorese out ofthe gel and onto the solid supportmembrane

How to confirm that proteins are transferred on nitrocellulose membrane ??

Staining gels by CBB No bands observed

Staining nitrocellulose membrane by Ponceau S dye

Orange-pink colour bands observed

Staining filter paper by Ponceau S dye

No bands observed

Not remaining on gel or transferred more to filter paper (below membrane)

Filter paperGelMembraneFilter paper

Check by staining

After transfer

Membrane is washed in a Tris buffer salineTween 20 solution (TBST).

Block membrane in non-fat dried milksolution. Usually 5% w/v. Preventsunwanted binding of antibodies tomembrane for 1 hr

Washing with TBST to remove blockingsolution

Third step

Primary antibody incubation step

The primary antibodies which specifically recognize the proteins of interest are used.

Primary Antibodies

After blocking membrane, add primary antibodies inconcentrations recommended by manufacture or indilution series if unknown onto the membrane.

Incubate overnight at 4oC or 2 hours at room temperature(only if it’s a good antibody).

Antibodies

Proteins that bind to specific epitopes on specific proteins.

Two main types used in western blotting Monoclonal

Polyclonal

Monoclonal are generally produced from mice and polyclonal are produced from rabbit or goat but other animals also produced polyclonals.

Antibodies for western blotting

Monoclonals are to one epitope while polyclonals

are to many epitopes on the protein of interest. If

epitope changes upon denaturing of the protein it

will not recognize the protein

Fourth step

Remove primary antibody by washing with TBST

Secondary antibody incubation step.

Use of secondary antibody which recognizes the

primary antibody used in the third step.

Secondary Antibodies

Wash at least three times with TBST for 5-10 minutes each

Added antibodies against the animal that the primary antibodies were made in.

These antibodies are also conjugated with enzymes such as horseradish peroxidase

The secondary antibodies are added to the membrane and incubated at room temperature for one hour.

Fifth step

Visualization step

Making the antigen-antibody complex visible (staining)

Autoradiography (radioactive P / chemiluminescence)

Avidin-biotin complex

Fluoresence method.

Detection of protein of interest

After secondary antibody incubation wash with TBST as before.

Add enhance chemiluminance reagents (commercially available) that have substrates for HRP that gives a product that gives off light energy. Capture the chemiluminescenceliberated on an X-ray film

After developing the x-ray film bands will appear.

or for detection – colored product

The location of the antibody isrevealed by incubating it with acolorless substrate that theattached enzyme converts to acolored product that can be seenand photographed

In brief western blot procedure

Advantages of western blotting

Western blot analysis can detect one protein in a mixtureof any number of proteins while giving you informationabout the size of the protein.

This method is, however, dependent on the use of a high-quality antibody directed against a desired protein.

This antibody is used as a probe to detect the protein ofinterest.

Detects proteins and estimates their molecular weight.

Detects changes in phosphorylation and lipid-modifications.

Used to detect changes in protein expression.

Application

1.The confirmatory HIV test

2.Western blot is also used as the definitive test for

Bovine spongiform encephalopathy (BSE(

WESTERN BLOT analysis