Protein blotting refers to transferring and immobilizing the proteins on a solid membrane support. The immobilized proteins, which are accessible to specific detection agents such as probes and antibodies, can be used for the detection and quantification of specific proteins as well as N-terminal sequencing.
The protein blotting technique was first introduced by Towbin et al., in 1979 (Towbin et al., 1979). The original procedure of protein blotting involved the electrotransfer of proteins from polyacrylamide gel to a nitrocellulose membrane, and subsequent detection using a protein-specific antibody. At present, protein blotting techniques especially western blot are routinely used by the researcher for the analysis of proteins.
Several protein blotting methods are available, of which western blotting (transferring of fractionated proteins from acrylamide gel to membrane) is the most common technique for the analysis of protein samples.
Depending on how the protein sample is transferred to the membrane, the protein blotting methods can be:
1. Manual spotting: A liquid solution of protein is manually spotted to the membrane (nitrocellulose or PVDF) and then allowed to dry.
2. Dot blot/Slot blot: This protein blotting technique uses vacuum-based microfiltration to immobilize protein onto the membrane. A special apparatus is needed for this.
3. Western blotting: Western blotting involves transferring protein from polyacrylamide gel to a membrane using an electric field. It is the most common technique to analyze protein in most laboratories.
Immobilized proteins can be used for
- N-terminal sequencing
- Mass spectrometry
Towbin et al., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76(9), 4350-4354. PMID-388439; Full-Text Links: PNAS, PMC411572