Western blotting uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane. The membrane can then be further processed with antibodies specific for the target of interest, and visualized using secondary antibodies and detection reagents.
Dot blot is a technique for detecting, analyzing and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate. Concentration of proteins in crude preparations (such as culture supernatant) can be estimated semiquantitatively by using the dot blot method if you have both purified protein and specific antibody against it.
(using primary antibody)
General procedure and tips for ELISA assay requiring a secondary conjugated antibody. For accurate quantitative results, always compare signal of unknown samples against those of a standard curve. Standards (duplicates or triplicates) and blank must be run with each plate to ensure accuracy.
A sandwich ELISA measures antigen between two layers of antibodies (capture and detection antibody). The target antigen must contain at least two antigenic sites capable of binding to antibodies. Monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonal antibodies recognize a single epitope that allows quantification of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. Sandwich ELISAs remove the sample purification step before analysis and enhance sensitivity (2–5 times more sensitive than direct or indirect). Sandwich ELISA procedures can be difficult to optimize and tested match-paired antibodies should be used. This ensures the antibodies are detecting different epitopes on the target protein and do not interfere with the other antibody binding.
Tagged fusion protein isolation with affinity gel
Sepharose-4B covalently attached with monoclonal antibody of certain protein tags are used to isolate fusion protein with such tags.
