Western blotWestern blot

Introduction

� A technique for detecting specific proteins byusing labeled antibodies. Also calledimmunoblotting. SDS-PAGE technique is aprerequisite for Western blot.

� This method was originated from the laboratory of� This method was originated from the laboratory ofGeorge Stark from Stanford

�Was first performed by Towbin et. al ( 1979 )

�Name was given by W. Neal Burnette – hassimilarity with Southern Blot

Why we do a Western Blot ?

1. To see if a specific protein of interest is present in asample

2. To compare the amounts of protein of interestamong different samplesamong different samples

3. To see if the state of a protein changes underdifferent biological conditions ( eg in disease, stressetc ).

Western blot involved three main steps :

1. Protein transfer and staining

2.Blocking and addition of antibodies2.Blocking and addition of antibodies

3.Visualization

Colours detected on film

Protein transfer –based on the surface area of the gel exposed to the electrical panel

Tank transfer Semidry transferTank transfer Semidry transfer

Note : Please use powder – free gloves when manipulating blotting papers, membrane and gels.

Equipment +Reagants

Tank transfer/Semidry transfer apparatus

Transfer buffer

Membrane

Blotting papers andsponge

SDS gel containing

protein samples

Transfer buffer1 ) Towbin buffer ( Tris –glycine-methanol)2 ) cyclohexylaminopropanesulfonic acid (CAPS)3 ) Tris - Borate

spongeHigh purity distilled water

Types of membrane

Membranes Description

Polyvinylidene diflouride ( PVDF ) ** Specifically used for protein sequencing** Have high retention – proteins will bind

tightly

Nitrocellulose ** Often used for western blot** Produce lighter background than PVDF** Produce lighter background than PVDF

Nylon ** Seldom used due to difficulties with staining

** More commonly used with nucleic acid transfer

Tank transfer protocol

Cut the SDS page gel, blotting paper and membrane to the appropriate size

Soak the membrane in H2O (methanol for PVDF ). Do slowly to prevent uneven wetting of membrane and formation of bubbles

Transfer the membrane to the transfer buffer. Allow to equilibrate for 5 minutesminutes

Assemble the transfer sandwich.

Place the transfer cassette into the tank.

Transfer according to the times and currents recommended by manufacturer of the transfer apparatus.

Place the lid on the transfer unit. ( Black is –ve cathode while red is +ve anode )

Fill the transfer tank with appropriate transfer buffer until completely covers electrode panels.

Mark the side of membrane facing the gel using soft lead pencil.

After finish, rinse membrane 3 x, 5 mins each with high purity water.

Stain the membrane with desired staining solution. Or store the membrane dry in the dark between blotting paper.

Tank Transfer unit

Semidry transfer protocol

Measure SDS-PAGE gel. Cut transfer membrane and six sheets of blotting paper to the same size of the gel

Presoak a membrane in H2O (methanol if PVDF ).

Soak the membrane and 6 pieces of blotting papers to transfer buffer . Allow to equilibrate for 5 minutes.equilibrate for 5 minutes.

Assemble the transfer sandwich

Transfer according to the times and currents recommended by manufacturer of the transfer apparatus.

Mark the orientation of membrane facing the gel and proceed with staining.

When the transfer is complete, turn off the power. Remove the membrane with a pair of forceps, rinse in deionized water.

Difference….

Tank transfer Semidry transfer

A sandwiched of gel, membrane and blotting paper are suspended vertically

A sandwiched of gel, membrane and blotting paper are placed horizontally

Litres of transfer buffer are needed Less much buffer are used

High voltage are required Less voltage required

Extended period of time in protein transfer

Less time in protein transfer

Type of staining solution

Stain Min. amount detect

Nitrocellulose

PVDF Nylon Comments √ / X with

Western Blot

Ponceau S 1 µg + + + Reversible √

CoomassieBlue

1 µg + + + PermanentHigh

background

√

India Ink 100 ng + + - Permanent -

Colloidal Gold

3 ng + + - Permanent X

Equipment + Reagants

Staining solution

Blotted membrane High purity

Soft lead pencil or

indelible ink

Shaker

membrane High purity distilled water

indelible ink

Staining membrane-bound proteins with Ponceau S

Submerge the membrane in Ponceau S solution with gentle agitation for 5 mins.

Decant the stain. Rinse the membrane several times with H2 O until the protein

bands visible.

Use soft lead pencil or indelible ink to mark the major protein spots and molecular-Use soft lead pencil or indelible ink to mark the major protein spots and molecular-

weight markers.

Rinse the membrane with H2 O, gentle agitation until the stain is removed

Proceed to the next step ( blocking and addition of antibodies )

Radiolabeled antibodies

Biotinylated antibodies

How to recognize the specific protein on transfer membrane?How to recognize the specific protein on transfer membrane?How to recognize the specific protein on transfer membrane?How to recognize the specific protein on transfer membrane?

Enzyme -linked antibodies ( Horseradish peroxidase or Alkaline phosphatase )

• A image on a x-ray film or nuclear emulsion produced by the pattern of decay emissions from distribution of a radioactive substance

Autoradiography

• Emission of light as the result of a Chemiluminescence

Methods to detect the presence of desired proteinsMethods to detect the presence of desired proteinsMethods to detect the presence of desired proteinsMethods to detect the presence of desired proteins

• Emission of light as the result of a chemical reactionChemiluminescence

• Measure the emission of coloursquantitatively Colorimetric

Equipments + Reagants

Blocking solution

e.g. 2% Bovine serum albumin ( BSA ), non fat dry milk, serum

1° and conjugated 2 ° antibodies

Blocking and addition of antibodies

1. Blocking the membrane by incubating the membrane in blocking solution

2. Incubate the membrane with 1° antibody – target to protein of interest

3. Wash off unbound 1°antibody with wash solution, 3x, 5 minutes each

6. Proteins identified by chromogenic or luminescent visualization of the protein-

antibody complex bound to the membrane.

4. Incubate with enzyme-linked antibody – will bind specifically to 1°antibody

5. Rinse the membrane with blocking solution, 3x, 5 minutes each

Chromogenic system

Enzymes Substrate Reaction/Detection Pro/Con

Horseradishperoxidase(HRP) -based

4-chloro-1-napthol( 4CN )

Oxidized substrate to form purple precipitate

Not very sensitive

3,3’-diaminobenzidine(DAB/NiCl2 )

Forms dark brown precipitate

Sensitive but carcinogenic

3,3’,5,5’-tetramethylbenzid

ine(TMB)

Forms dark purple stain

Stable, less toxic than DAB/NiCl2

Alkaline phosphatase(AP) - based

5-bromo-4-chloro-3-indolyl phosphate

(BCIP)/Nitrobluetetrazolium (NBT)

Dark blue-gray stain results

Sensitive

Luminescent system

Enzymes Substrates Detection/Reaction Pro/Con

HRP-based Luminol/H2O2/p-iodophenol

Oxidized luminol gives off blue light,p –

iodophenol increases light output

Sensitive, convenient

AP-based Substituted 1,2-dioxetane-phosphatase (eg Lumi-Phos530, Lumigen-

PPD )

Dephosphorylated gives off light

Sensitive

Example of blotted membrane

Having some problems???

Problems Cause Solution

1 ) Poor protein transfer

1 ) Type of gel used to separate protein 2 ) Gel thickness

1 ) Gel thickness up to 1.5mm

2 ) Transfer membrane blank and little or no protein left in the gel after transfer

1 ) Electrode were connected in reversed order2 ) Membrane were placed on the wrong side of gel

1 ) Make sure the orientation of electrode and membrane in correct order

3 ) Blank spots on 1 ) Cause by air bubbles – 1 ) Carefully remove air 3 ) Blank spots on membrane4 ) Presence of bright white spots within protein bands on the membrane

1 ) Cause by air bubbles –block the flow of current

1 ) Carefully remove air bubbles by rolling a pipette or glass rod across the surface of the membrane2 ) Lift membrane carefully to release air bubbles and replace it w/o shifting the position of either gel or membrane

Having some problems???

Problems Cause Solution

5 ) Protein have transferred efficiently but stained membrane has blurred bands or swirled pattern

1 ) Insufficient contact between the gel and membrane during protein transfer

1 ) Ensure close contact between the gel and membrane2 ) Held the transfer sandwich firmly together

6 ) High background stain

1 ) Insufficient blocking or non-specific binding of the 1° or 2° antibody

1 ) Switch to another blocking solution2 ) Decrease the 1° or 2° antibody 2 ) Decrease the concentration of primary antibody

How to make the transfer sandwich ( tank transfer )How to make the transfer sandwich ( tank transfer )How to make the transfer sandwich ( tank transfer )How to make the transfer sandwich ( tank transfer )

1. Place one side of the transfer cassette in a tray of transfer buffer

2. Place a foam sponge onto cassete and squeeze out any bubbles

3. Place one sheet of blotting paper on top of sponge. Make sure it wet evenly and completely

4. Place the SDS gel onto the surface of the blotting paper

5. Place the transfer membrane on top of the gel

6. Layer another piece of blotting paper on top of transfer membrane

7. Then, place another foam sponge onto the cassette.

8. Close the cassette and snap it shut

How to make the transfer sandwich ( semidry tank transfer )How to make the transfer sandwich ( semidry tank transfer )How to make the transfer sandwich ( semidry tank transfer )How to make the transfer sandwich ( semidry tank transfer )

1. Stack 3 pieces of blotting paper onto anodic electrode panel of the transfer unit. Remove bubbles if any.

2. Place the transfer membrane onto the surface of the blotting papers.

3. Lay the SDS-PAGE gel on the top of membrane.

4. Put another 3 pieces of blotting paper on top of the gel.

5. Then, place the cathode panel onto the transfer stack