Protein Reverse Staining: High-Efficiency Microanalysis of Unmodified Proteins Detected on Electrophoresis Gels

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

“…The introduction of imidazole for zinc staining overcame this problem in SDS-PAGE applications [101] and was further modified for application to non-SDS gels [102]. These marked improvements to zinc staining resulted in a large number of studies aiming to further enhance the method and demonstrate that proteins within zinc stained gels were still biologically active, could be recovered with high yields and were compatible with MS technologies [103][104][105][106][107][108][109][110]. Since this process leaves proteins presumably untouched, significant effort was invested in highlighting its qualitative potential and studies examining its quantitative capacity have been less prevalent.…”

“…The consensus for zinc-imidazole-stained gels Yes [20], [73], [157,158] 500 ng A. thaliana total protein SR, MeOH/HAc fixation, stain from 3 h to overnight, destain in MeOH/HAc, water washed. was also to take images, against a black background in this case, usually using a Polaroid camera or an automated imager, and follow with analysis [20,100,101,104,107,108,243]. Fluorescence posed a different challenge in terms of detection given the specific excitation and emission requirements of different fluorophores.…”

Molecular cloning, gene expression analysis, and recombinant protein expression of novel silk proteins from larvae of a retreat-maker caddisfly, Stenopsyche marmorata

“…S1), the gel bands of Smsp-3 and Smsp-4 stained by the reverse staining method [7] were excised, destained, and crushed. The whole proteins were eluted from the gel fragments by formic acid/water/2-propanol (1:3:2 v/v/v) [8] and analyzed using a MALDI-TOF mass spectrometer, TOF/TOF 5800 System (AB SCIEX) using a sinapic acid matrix.…”

“…SDS-PAGE analysis (20% gel) of the P3′ fraction of the major silk proteins extracted from the silk glands [5,6]. The proteins were stained by the reverse staining method [7] for the subsequent mass spectrometry analysis. [13,14] and the N-terminal amino-acid sequence of Smsp-3 from Stenopsyche marmorata.…”

“…S1), the gel band of Smsp-3 stained by the reverse staining method [7] was excised, crushed, and digested by trypsin in the gel, according to the modified protocol [20] based on the standard method [21], as follows: ・ The gel band was excised and crushed, and the gel pieces were washed and destained with destaining solution (25 mM Tris-HCl, 192 mM glycine, pH 8.3) several times. ・ Dehydration by soaking in 100 mL of acetonitrile and evaporation of the solvent were performed.…”