Immunodetection of proteins on a Western blot requires the use of expensive antibodies, but poor or incomplete transfer would result in wasting these antibodies. A Western transferred blot can be checked for success of protein transfer with a protein stain and therefore prevent wasting expensive antibodies. Various options for protein stains for Western blot are available, including, but not limited to, Ponceau S, Amido black 10B, Coomassie brilliant blue R250, India ink or colloidal gold. In addition to checking success and quality of Western transfer, staining blots is also used for semi-quantitative protein estimation. In addition, checking the blot with a stain gives a rough idea to a researcher that the desired protein is present (based on size, mobility etc) and whether to go for immunodetection especially when expensive and limiting amounts of antibodies are available.
Essentially all stains that are used for staining gels can be used to stain the blot membranes; however their sensitivity may vary in gel and on blot. In addition the purpose of staining a blot membrane is different than staining the gel. For example, Ponceau S stain is preferred stain as it is rapid and reversible. It can be washed away and thus does not interfere with immunodetection process.
One should also take into account the type of membrane used for transfer when selecting a protein stain. Not all stains are compatible with all membranes. The three widely used membranes for protein blotting are nitrocellulose, nylon and polyvinylidene difluoride (PVDF).
The most commonly used stains for protein detection on blot membranes are as follows:
Anionic dyes (Ponceau S, Fast green FCF, Amido Black 10 B, Coomassie brilliant blue R250)
Ponceau S and fast green are rapid and reversible proteins stains that can be removed easily and does not interfere with immunodetection process. The Ponceau S is most widely used and its sensitivity is around 100 ng1. Coomassie brilliant blue R250 and amido black 10B are more sensitive than Ponceau S2. However with Coomassie brilliant blue R250 the background staining is high and also this stain is not removed easily. Coomassie Brilliant Blue also interferes with immonodetection process. Amido Black 10B can be removed easily but it may interfere with downstream immunodetection process. Amido Black 10B is most commonly used as post-antibody stain.
Colloidal gold or silver
This staining is based on high-affinity binding of colloidal gold or silver to proteins on blot via electrostatic adsorption. The protein bands are stained red-purplish color when colloidal gold is used or dark grey when colloidal silver is used. The sensitivity of colloidal gold or silver is 1 ng 3. This staining takes longer time, is not easily reversible, and is not compatible with downstream immunodetection process.
Fluorescent stains
Commercially available fluorescent stains are permanent and offers high sensitivity in range of 2-8 ng. Although the staining is permanent, fluorescent stains are compatible with colorimetric or chemiluminescence immunodetection method.
Stain-free detection
2, 2, 2-Trichloroethnaol is added to the polyacrylamide solution before casting a gel4. The principle behind stain free method is that the tryptophan amino acids in protein undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in visible range (300 nm). The fluorescent signal on blot can be detected by a CCD camera
References
1. Aebersold, R. et al (1987). Proc. Natl. Acad. Sci. USA, 84:6970
2. Harper, S. and Speicher, D.W (2001). Curr Protoc Protein Sci. Chapter 10: Unit 10.8. doi: 10.1002/0471140864.ps1008s00.
3. Moeremans, M. et al (1985). Anal Biochem. 145(2):315-21
4. Ladner, C. L. et al (2004). Anal Biochem. 326(1):13-20
