1. Western Blotting

2. 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.

3. Introduced by Towbin, et al
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.

4. Flow chart of Western blotting
Electrophoresing the protein sample
Assembling the Western blot sandwich
Transferring proteins from gel to nitrocellulose paper
Staining of transferred proteins
Blocking nonspecific antibody sites on the nitrocellulose paper
Probing electroblotted proteins with primary antibody
Washing away nonspecifically bound primary antibody
Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitate
Photographing the immunoblot

5. SDS polyacrylamide-gel electrophoresis (SDS-PAGE)

6. SDS-PAGE 1) Thin gel separates molecules on the basis of size.
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.

8. 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).

10. Components for SDS-PAGE
Tris buffer to provide appropriate pH
SDS (sodium dodecyl sulfate)
detergent to denature proteins and give them a negative charge.
Glycerol to make samples sink into wells
Blue dye to visualize samples as gel is run.

11. 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.

12. 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.

13. 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.)

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

16. 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).

17. Western blotting
Detects proteins and estimates their molecular weight.
Detects changes in phosphorylation and lipid modifications.
Used to detect changes in protein expression.

18. 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.

19. 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.

20. A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease'). Some forms of Lyme disease testing employ Western blotting

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