Western blots are used to confirm the identity of antigens by separating proteins by size using gel electrophoresis, transferring them to a membrane, and detecting them using antibodies. The proteins are first separated by SDS-PAGE gel electrophoresis, then transferred to a membrane where they are probed with patient sera or primary antibodies. Secondary labeled antibodies are then used to detect the presence and molecular weights of antigens recognized by the primary antibodies or patient sera.

1. Western Blot
Sunil Bhandari

3. • Western blots are labor intensive and expensive but provide a
method of confirming the identity of a reactive antigen
• Western blots are done by electrophoresis of antigen (sample
protein) by sodium dodecyl sulfate ( polyacrylamide gel
electrophoresis (PAGE)
• The proteins in a mixture are separated by size in the gel and
are transferred from the gel to a solid paper like substrate
often called a membrane.
• The membrane is incubated with patient sera and the presence
of patient antibodies is detected by subsequently incubating
the membrane with labeled secondary antibodies.
• The specificity of the western blot lies in its ability to demonstrate
the molecular weights ( of the proteins recognized by patient sera

4. Principle:
• Western blotting is a method where a labelled antibody against
particular protein is used to identify the desired protein, so it is a
specific test. Western blotting is also known as immunoblotting because
it use antibodies to detect the protein.
• Western blot is performed by using polyacrylamide gel
electrophoresis (SDS-PAGE) which allows protein samples to be
separated and transferred to a solid support such as nitrocellulose
membrane.
• This solid support can absorb the protein and keep its biological
activity unchanged.
• The protein transferred solid support membrane is called a blot and
is treated with a protein solution to block the hydrophobic binding
site on the membrane.

5. • The membrane is treated with the antibody (primary antibody)
of the target proteins. Only the proteins to be studied can
specifically bind to the primary antibody to form an antigen
antibody complex.
• The primary antibody treated membranes are treated with a
labeled secondary antibody after washing. After treatment, the
labeled secondary antibody that binds to the primary antibody
forms an antibody complex that can indicate the location of the
primary antibody, i e the location of the protein being studied.

6. Process of western blot