Western blot (SDS–PAGE → semi-dry transfer → immunodetection) — step-by-step protocol Overview: Western blotting ( Immuno-Blotting ) is a technique which allows to detect specific proteins ( antigens ) via the interaction with a corresponding antibody . Therefore, proteins which were first separated by their molar mass on a polyacrylamide gel are transferred to a nitrocellulose or polyvinylidenfluorid ( PVDF ) membrane by applying an electric field. The membrane is then exposed to a primary antibody solution where the antibody binds solely to the desired protein. Next, the membrane is incubated with a secondary antibody which subsequently binds to the first antibody. The secondary antibody carries an enzyme which catalysis a chemical reaction when exposed to a detection solution resulting in a fluorescent signal that can be measured. Western blotting allows to detect protein amounts below the nanogram level Material and Chemicals you will need: Rotib...
Comments
Post a Comment