An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

Abstract

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix-assisted laser desorption/ionisation-time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post-staining gels subjected to labeling with other fluorophores such as Schiff's base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post-staining gels after detection of glycoproteins using the green fluorescent Pro-Q Emerald 300 glycoprotein stain or detection of beta-glucuronidase using the green fluorescent ELF 97 beta-D-glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.

A fluorescent natural product for ultra sensitive detection of proteins in one‐dimensional and two‐dimensional gel electrophoresis

“…The detection limit of protein in gels stained with SYPRO Ruby was in the order of 4 ng to 500 pg (Fig. 2a), which was in keeping with that found in other studies [2,8,9,22], and was an order of magnitude less sensitive than Lightning Fast. The high degree of speckling on the SYPRO Ruby gel was a concern and was therefore repeated but produced the same result.…”

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

“…Ruthenium (II) tris (bathophenanthroline disulfonate), RuBPS (also known as RuBPSA and RuBTS) was introduced as an economical alternative to SR in 2000 [162]. Although it was suggested that RuBPS delivered superior sensitivity in comparison to SR [163], it was later demonstrated that there was no quantitative advantage over the original or optimized SR formulation [164]. It is, however, definitely more cost-effective and has thus been used in various proteomic investigations [165][166][167][168][169][170].…”

“…It is, however, definitely more cost-effective and has thus been used in various proteomic investigations [165][166][167][168][169][170]. It has been shown that the limit of sensitivity of RuBPS is approximately 10 ng protein/band (broad-range MW standards, Bio-Rad), as reported previously [164] and determined qualitatively from published data [163]; however, a more detailed evaluation of RuBPS staining indicated this sensitivity threshold to be much lower, at ∼2 ng protein/band (CA, soybean trypsin inhibitor, OVA, albumin, PhosB) [168]. Similarly to SR, the LDR for RuBPS was between 2 and 2,500 ng for BSA [168].…”

An Improved Protocol for Better Detection of Protein Using 8-Anilino-1-naphthalenesulfonate

“…The staining method was essentially according to Berggren et al 13 2-D gels were fixed after electrophoresis using 400 mL of a 10% MeOH/7% HAc solution for 1 h; fixing was not required for 1-D gel before staining. 1-D electrophoretic gels were placed into 50 mL (300 mL for 2-D gel) of a SYPRO Ruby staining solution for no less than 3 h. The gels were then rinsed briefly in a 10% MeOH/7% HAc solution for 30 min.…”