Protein Blotting
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Protein Blotting
Related Applications
Protein blotting has been used in research laboratories since the 1970s and refers to the process of immobilizing solubilized proteins on a membrane for further analysis.
Although analysis can be as simple as confirming the presence of a specific protein in a complex sample, recent advances in detection reagents and imaging systems have led to the evolution of protein blotting as a technique for precise protein quantitation.
The preliminary separation of proteins on a gel provides data on the various molecular weights as well as the structural integrity of proteins in a complex sample. In addition, other analyses may be performed, such as the detection of protein interactions following co-immunoprecipitation, or identification using techniques such as N-terminal sequencing or mass spectrometry.
The simplest blotting procedure is known as dot blotting or slot blotting; the direct application of a protein solution onto a membrane, usually with the aid of a vacuum pump. From these beginnings, Western blotting emerged in 1979. Western blotting is now the most widely used and information-rich protein blotting technique in which proteins are electrophoretically separated in a polyacrylamide gel prior to electrotransfer onto a membrane.
A family of related protein blotting techniques has evolved to include Eastern blotting for the detection of post-translational modifications, Far Western blotting for non-antibody protein: protein interactions, and Far Eastern blotting for lipid detection. Southwestern and Northwestern blotting are recent additions to the repertoire of blotting techniques, used for the detection of protein interactions with DNA and RNA, respectively.
The earliest methods for detecting blotted proteins were based on antibodies labeled with radioisotopes, such as 32P or 35S. Although radioisotopes are sensitive, safety issues gradually led to their replacement by antibody probes conjugated with an enzyme for enhanced chemiluminescence (ECL) or chemifluorescence detection, or directly with fluorophores for fluorescence detection. In addition, using multiple fluorophores that emit light at distinct wavelengths allows the simultaneous detection of several proteins on a single blot in a technique known as multiplexed detection.
