Comparative advantages of imidazole-sodium dodecyl sulfate-zinc reverse staining in polyacrylamide gels.

Polyacrylamide gel electrophoresis (PAGE), which is widely used to assess protein purity and molecular weight [1], is best combined with a highly sensitive, reproducible, inexpensive, rapid, and easy to perform protein staining/detection method. Coomassie brilliant blue (CBB)1 staining is a popular method, known for its simplicity, economy, and compatibility with downstream analysis but relatively poor sensitivity (about 50 ng protein/band), low aYnity for acidic proteins, variability in background staining, and lack of reproducibility. The silver staining procedure is sensitive but relatively tedious and is not speciWc for proteins [2,3]. Rutheniumcomplex-based SYPRO Ruby Xuorescent staining is protein speciWc and as sensitive as silver staining. It has a broad linear dynamic range and a simple stain/destain protocol and is compatible with subsequent mass spectrometry or Edman sequencing [4,5]. Imidazole–SDS– zinc reverse staining is reportedly more sensitive than CBB staining [6] and approximately as sensitive as silver staining [2]. In this method a white, insoluble imidazole– zinc complex forms on the surface of gels as a background against which proteins, complexed with SDS during pretreatment, appear as unstained, transparent bands [7]. In the study reported here, a comparison of the imidazole–SDS–zinc, SYPRO Ruby, silver, and CBB staining methods was made. Imidazole–SDS–zinc reverse staining, with minor modiWcations of the original method [6,8], was shown to best meet all the optimal criteria (as stated above), while allowing sensitive, reproducible detection and sample recovery.