Improved conditions for fluorescent staining of proteins with 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid in SDS‐PAGE

Abstract

A simple and sensitive fluorescent staining method for the detection of proteins in SDS-PAGE, namely IB (improved 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) stain, is described. Non-covalent hydrophobic probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid was applied as a fluorescent dye, which can bind to hydrophobic sites in proteins non-specifically. As low as 1 ng of protein band can be detected briefly by 30 min washing followed by 15 min staining without the aiding of stop or destaining step. The sensitivity of the new presented protocol is similar to that of SYPRO Ruby, which has been widely accepted in proteomic research. Comparative analysis of the MS compatibility of IB stain and SYPRO Ruby stain allowed us to address that IB stain is compatible with the downstream of protein identification by PMF.

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

“…It was reported that SR again yielded a LLD of 1-2 ng (SDS-6H marker proteins; Sigma) and a LDR of 2-500 ng [158]; little inter-protein variation was suggested by the LDR plot shown. Also, MALDI-ToF-MS analysis of standard proteins (BSA and CA) displayed sequence coverage between 29-34% and 48-58%, respectively (for protein loads between 4 and 64 ng) [157]. A comparison of this and the previous studies suggests that the LLD values are consistent.…”

“…Independent studies using the SR staining method detailed previously [20] were also carried out [157,158]. It was reported that SR again yielded a LLD of 1-2 ng (SDS-6H marker proteins; Sigma) and a LDR of 2-500 ng [158]; little inter-protein variation was suggested by the LDR plot shown.…”

“…Significant changes to the original staining method for BisANS have recently achieved comparable protein detection sensitivity to that seen with SR [157]. The original BisANS staining protocol according to Horowitz and Bowman [184] washed gels in water, stained with 20 μm BisANS (in water), washed in 2 M KCl, and rinsed briefly in water.…”

“…The original BisANS staining protocol according to Horowitz and Bowman [184] washed gels in water, stained with 20 μm BisANS (in water), washed in 2 M KCl, and rinsed briefly in water. The method developed by Cong et al [157] differed significantly; gels were first fixed in EtOH/HAc, washed in water, stained with 0.0002% BisANS in EtOH/HAc and rinsed briefly with water before imaging. The LLD of standard protein markers (SDS-6H2; Sigma) was 1 ng/band and the LDR between 1 and 250 ng; both values were comparable to those obtained using SR [157].…”

“…The method developed by Cong et al [157] differed significantly; gels were first fixed in EtOH/HAc, washed in water, stained with 0.0002% BisANS in EtOH/HAc and rinsed briefly with water before imaging. The LLD of standard protein markers (SDS-6H2; Sigma) was 1 ng/band and the LDR between 1 and 250 ng; both values were comparable to those obtained using SR [157]. MS analysis (MALDIToF) showed similar sequence coverage for both BisANS and SR for two standard proteins.…”