PRESENTED BY

Raja Muhammad MunsifMudasar Majeed Raja Qadir IqbalKhawaja Waqas AhmedShahzad Naseer Awan

UNIVERSITY OF AZAD JAMMU AND KASHMIR MUZAFFARABADDEPARTMENT OF BOTANY

WESTERN BLOTTINGDefination

The Western Blot is an analytical technique used to

detect specific proteins in a given sample of tissue

homogenate or extract.

Western blotting is an immunoblotting(protein

detection) technique used for the separation, to

assess the presence, amount and molecular- weight

of proteins in cellular or tissue extracts by using

antibodies.

Western Blotting – Used to detect protiens

Southern Blotting - Used to detect DNA

Northern Blotting - Used to detect RNA

TYPES OF BLOTTING TECHNOQUES

PRINCIPLE OF WESTERN BLOTTING

Western blotting is an Immunoblotting technique whichrely on the specificity of binding between a molecule ofinterest and a probe to allow detection of the molecule ofinterest in a mixture of many other similar molecules.

In Western blotting, the molecule of interest is a proteinand the probe is typically an antibody raised against thatparticular protein.

The SDS PAGE technique is a prerequisite for Westernblotting

THE PROCEDURE INCLUDES

Tissue preparation Gel electrophoresis Transfer Blocking Detection Analysis

TISSUE PREPARATION Samples may be taken from whole tissue or from cell

culture. In most cases, solid tissues are first broken downmechanically using a blender.

It should be noted that bacteria, virus or environmentalsamples can be the source of protein and thus Westernblotting is not restricted to cellular studies only.

Assorted detergents, salts, and buffers may be employed toencourage lysis of cells and to solubilize proteins.

Tissue preparation is often done at cold temperatures toavoid protein denaturing.

GEL ELECTROPHORESIS The proteins of the sample are separated

using gelelectrophoresis. Separation of proteins may be byisoelectric point molecular weight, electric charge, or acombination of these factors.

The principle involved is the difference in theELECTROPHORETIC MOBILITIES of different proteins

SDS-PAGEThe SDS-PAGE is sodium dodesylsulphate

polyacrylamide gel electrophoresisSDS-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.

APPARATUS OF SDS-PAGE Casting Stand Clamping Frame Sample loading guide Glass plates Plastic comb Casting frame

PRINCIPLE OF SDS-PAGE

Proteins move in the electric field.

Their relative speed depends on the charge,

size, and shape of the protein

TRANSFERINGTransfer 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 apparatusNo transfer buffer chamberOnly has plates (runs for hours)

Submerge transfer apparatusContains a chamber filled with

transfer bufferElectrodes -Plate electrodes (runs for

hours) -Wire electrodes (runs

overnight)

TRANSFER THE PROTEIN FROM THE GEL TO THE MEMBRANETransfer of the proteins fractionated by SDS-PAGE to a solid support membrane (Western blotting) can be accomplished by electro blotting

RUNNING A TRANSFER

Similar to running a gelNegatively charged proteins run towards the anode end of the transfer apparatus

Always have nitrocellulose on the anode side to capture proteins.

PVDF MEMBRANEWe 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

AFTER TRANSFER

Membrane is washed in a Tris buffer saline Tween 20 solution (TBST). Block membrane in non-fat dried milk solution. Usually 5% w/v. Prevents unwanted binding of antibodies to membrane for 1 hrWashing with TBST to remove blocking solution

BLOKING The membrane has the ability to bind to proteins in this

case both the target and antibodies are proteins and so therecould be some unwanted binding.

Blocking of non-specific binding is achieved by placing themembrane in a dilute solution of protein - typically Bovineserum albumin(BSA) with a minute percentage of detergentsuch as Tween 20.

The protein in the dilute solution attaches to the membranein all places where the target proteins have not attached.Thus, when the antibody is added, there is no room on themembrane for it to attach other than on the binding sites ofthe specific target protein.

THIRD STEPPrimary antibody incubation step

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

PRIMARY ANTIBODIES

Proteins that bind to specific epitopes on specific proteins.Two main types used in western blottingMonoclonal Polyclonal

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 STEPSecondary antibody incubation step.

Use of secondary antibody which recognizes the primary antibody used in the third step. 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 peroxidase

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

FIFTH STEPVisualization step

Making the antigen-antibody complex visible (staining)

Autoradiography (radioactive P / chemiluminescence)

Avidin-biotin complex Fluoresence method.

ANALYSIS After the unbound probes are washed away, the

westernblot is ready for detection of the probes that are labeledand bound to the protein of interest.

Size approximations are taken by comparing the stainedbands to that of the marker loaded duringelectrophoresis.

The process is repeated for a structural protein, such asactin or tubulin that should not change between samples.

ADVANTAGES

While ELISA being a non specific test, Western blotting is amore specific test for detection of HIV.

It can detect one protein in a mixture of proteins while givinginformation about the size of the protein and so is more specific. 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 of interest. Detects proteins and estimates their molecular weight. Detects changes in phosphorylation and lipid- modifications. Used to detect changes in protein expression.

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