western blotting. western blotting/immunoblotting technique for detecting specific proteins...

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Western Blotting

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Page 1: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Western Blotting

Page 2: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Western blotting/Immunoblotting

Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies.

Allows to identify a particular protein of interest among many proteins in a sample.

Page 3: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis.

The specificity of the antibody-antigen interaction enables a single protein to be identified in the midst of a complex protein mixture.

Page 4: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Flow chart of Western blottingFlow chart of Western blotting

Electrophoresing the protein sampleElectrophoresing the protein sample

Assembling the Western blot sandwichAssembling the Western blot sandwich

Transferring proteins from gel to nitrocellulose paperTransferring proteins from gel to nitrocellulose paper

Staining of transferred proteinsStaining of transferred proteins

Blocking nonspecific antibody sites on the nitrocellulose paperBlocking nonspecific antibody sites on the nitrocellulose paper

Probing electroblotted proteins with primary antibodyProbing electroblotted proteins with primary antibody

Washing away nonspecifically bound primary antibodyWashing away nonspecifically bound primary antibody

Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitateand formation of a diaminobenzidine (DAB) precipitate

Photographing the immunoblotPhotographing the immunoblot

Page 5: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

SDS polyacrylamide-gel electrophoresis (SDS-PAGE)

Page 6: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

SDS-PAGE

Stands for “SDS Polyacrylamide Gel Electrophoresis

1) Thin gel separates molecules on the basis of size. 2) Negatively charged molecules are loaded into wells in a gel that is submerged in buffer. 3) Electrical current is applied to the gel, causing the molecules to move toward the positive electrode (the anode,

usually marked red). 4) Smaller molecules move faster through the gel than larger molecules Thus, smaller molecules will be found at the bottom of the gel

and larger molecules will be found at the top of the gel.

Page 7: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies
Page 8: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

SDS-PAGE

• Some differences from agarose gel electrophoresis:

1) SDS-PAGE is usually used to separate protein molecules;

Agarose gels are usually used to separate DNA molecules.

2) Proteins must be coated with SDS before being loaded in the gel. SDS is a negatively charged detergent that gives the proteins a negative charge and denatures them (makes them linear instead of 3-D).

Page 9: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies
Page 10: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Components for SDS-PAGE

Tris buffer to provide appropriate pHSDS (sodium dodecyl sulfate)

detergent to denature proteins and give them a negative charge.

Glycerol to make samples sink into wellsBlue dye to visualize samples as gel is

run.

Page 11: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Western blot step-by-step

• 1- The first step in a Western blotting procedure is to separate the macromolecules using gel electrophoresis.

• 2- Size separation of the proteins in the mixture by Polyacrylamide Gel Elecrophoresis (PAGE).

• 3- The separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene fluoride (PVDF) membrane.

• 4- The membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the membrane.

Page 12: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

• 4- The transferred protein is complexed with an enzyme-labeled antibody as a probe.

• 5-An appropriate substrate is then added to the enzyme and together they produce a detectable product such as a chromogenic or fluorogenic precipitate on the membrane for colorimetric or fluorimetric detection,

• 6-The most sensitive detection methods use a chemiluminescent substrate that, when combined with the enzyme, produces light as a byproduct.

Page 13: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Why do a Western blot?

• 1) To see if a specific protein of interest is

present in a sample

• 2) To compare the amounts of a protein

of interest among different samples.

• 3) To see if the state of a protein

changes under different biological

conditions (e.g. in disease, stress, etc.)

Page 14: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Analysis of protein samples by SDS polyacrylamide-gel electrophoresis and Western

blotting

Page 15: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies
Page 16: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Applications

• 1- Western blot has been widely used in analyzing and identifying target proteins.

• 2- Western blot has also been used in clinical laboratories for assisting identification of certain antigen proteins (pathogen or biomarker).

Page 17: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Western blotting

• Detects proteins and estimates their molecular weight.

• Detects changes in phosphorylation and lipid modifications.

• Used to detect changes in protein expression.

Page 18: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Advantages

• While ELISA being a non specific test, Western blotting is a

• more specific test for detection of HIV.• It can detect one protein in a mixture of proteins while

giving• information about the size of the protein and so is more• specific.• Western blot test is referred to as the 'Gold Standard'• It also tells you how much protein has accumulated in

cells.

Page 19: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

Western Blot in Clinical Medicine

• The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIVinfected

• cells are separated and blotted on a membrane then, the• serum to be tested is applied in the primary antibody

incubation step;• free antibody is washed away, and a secondary anti-

human antibody• linked to an enzyme signal is added. The stained bands

then indicate• the proteins to which the patient's serum contains antibody.

Page 20: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

A Western blot is also used as the definitive test for Bovinespongiform encephalopathy (BSE, commonly referred to as

'mad cowdisease').

Some forms of Lyme disease testing employ Western blotting

Page 21: Western Blotting. Western blotting/Immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies

The virus is enveloped with different prThe virus is enveloped with different proteins.The detection of these proteins are useful in thedetection of the presence of the virus.Western blotting helps in the detection of these proteins.oteins.The detection of theseproteins are useful in thedetection of the presenceof the virus.Western blotting helps inthe detection of theseproteins.