This paper describes the use of a
ruthenium complex ((bis(
2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-
ruthenium-N-succidimyl
ester-bis(hexafluorophosphate), abbreviated below as
ASCQ_Ru) commercially available and chemically pure. This new
ruthenium complex
ASCQ_Ru brings an activated
ester, allowing the selective acylation of
amino acid side chain
amines for the post migration staining of
proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the
proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in
solution the best described pH for acylating
proteins with this
reagent is
phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered
ACN/water
solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7%
acetic acid/10%
ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on
protein molecular weight markers. We obtained a higher sensitivity than
SYPRO Ruby with a detection limit of 80 pg of
protein per well. However, contrary to
SYPRO Ruby,
ASCQ_Ru exhibits a logarithmic dependency on the amount of
protein. The dynamic range is similar to
SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration
ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole
protein extract from human colon
carcinoma cells lines HCT 116.
ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB,
SYPRO Ruby and Deep Purple.