Western blotting Western blotting Western blotting starts with separation of differently charged proteins via electrophoresis on polyacrylamide. The separated proteins are transferred to a sheet of nitrocellulose by placing the nitrocellulose directly on the polyacrylamide, and running a current perpendicularly through the combination. The nitrocellulose sheet is cut into strips. One strip is developed with a protein stain to identify the position of all the components. Unreacted antibodies are removed by washing following which the strip is incubated with fluorescent or radiolabeled antihuman antisera.
Positive spots indicate that the patient serum contains antibodies to the particular antigen.
This test is best known as the definitive screen for anti-HIV antibodies, but it is also widely used in research.