RAMA stain: A fast, sensitive and less protein-modifying CBB R250 stain

Abstract

SDS-PAGE and CBB staining are two of the most popular methods used for protein analysis. Although many reports that describe such staining methods have been published, these conventional protocols require several hours or days for staining and de-staining. In this study we describe a recently developed, fast and sensitive CBB staining method that utilizes the staining solution of RAMA that consists of the low-cost reagents: CBB R250, acetic acid, methanol and ammonium sulfate, and the destaining solution of water. Our method dose dependently detects 12 nanograms protein within 60 min and with a wide protein spectrum. Although the features of the dose-dependent relationship depend upon protein amounts and protein types, for most of the protein samples tested, a linear relationship was observed in the region from 12 to 330 ng. Moreover, through further washing, the detection sensitivity of protein is enhanced and reaches a maximum at 1.4 ng and then gradually decreases in the de-staining process. It has been shown recently through MS analyses that the sensitive colloidal CBB staining methods frequently result in artifactual methylations due to the strong acid and long contact during staining and the destaining processes. Such artifacts were reported to be reduced by the replacement of strong inorganic acid with acetic acid and because RAMA utilizes acetic acid and is in contact with the proteins for a short time during staining and de-staining, it is expected that in vitro artifacts will be reduced. Finally, MS analyses of RAMA-stained protein bands were revealed not to have been methylated.