High-eYciency staining of proteins on diVerent blot membranes

Immunodetection of proteins on blot membranes, Wrst developed by Towbin et al. [1], is the method of choice for identifying speciWc proteins after electrophoresis. Due to its high resolution, immunoprobing of blots from twodimensional polyacrylamide gels has been used extensively as a means of localizing speciWc proteins among hundred or even thousands of separated proteins [2]. Three major types of membranes are used for protein blotting: nitrocellulose, nylon, and polyvinylidene diXuoride (PVDF).1 Typically, before immunodetection, a blot is subjected to a general protein stain such as Amido black 10B [3], Ponceau S [3], Coomassie brilliant blue R-250 [4], India ink [5], or Colloidal gold [2,6]. Of the protein stains, Amido black, Ponceau S, and Coomassie blue are the least sensitive, with a detection limit of approximately 50 ng protein. India ink has an intermediate sensitivity of detection and takes approximately 4 h to complete. Colloidal gold stain has the highest sensitivity on PVDF blot membrane (1–2 ng) but requires several hours to complete. To be eVective, most of the above protein staining procedures are carried out under acidic conditions (e.g., acetic acid) and/or organic solvents (e.g., methanol). The eYciency of staining also varied from membrane to membrane. For example, it has been reported that because of high staining background, nylon membrane is not compatible with all of the above dyes [7]. Furthermore, nitrocellulose membrane cannot be used when high concentrations of organic solvents such as methanol are used [7]. The use of acid and/or organic solvents on blots can also cause problems for the detection of labile antigenic activity, enzymatic activity, protein–protein interactions, and protein–ligand interactions [8].