Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining

Abstract

The study of effects of several parameters on silver staining of proteins has led to the development of a staining method which is simple and reliable, requires only few stable solutions, and can be applied to all gel types such as sodium dodecyl sulfate (SDS) containing polyacrylamide or isoelectric focusing gels. It can be used for ultrathin layers (0.1-0.2 mm) or thicker slab gels (up to 3 mm). With this method not only proteins but also polypeptides ofmolecular weights as low as 2500 are detectable with high sensitivity. Comparison of isoelectric focusing and SDS-containing gels and a simple spot test on thin gel layers show that the detection sensitivity depends not so much on the type of proteins but rather on their structure. The redox properties of the gel are important for the staining mechanism.

The mechanism and regulation of deadenylation: Identification and characterization of Xenopus PARN

“…Ten milliliters of S100 (;400 mg protein) were loaded onto a 2+6 ϫ 26 cm column, packed with DEAE-Sephacel (Pharmacia), fitted with a flow adapter and washed at 1 mL/min with DB until A 280 reached baseline+ The column was eluted with a 180-mL gradient from 40 to 400 mM KCl in DB+ Fractions containing xPARN were identified by TLC-assay (see below) using 1 mL of every fifth fraction+ Active fractions were pooled, then diluted with one volume of DB (;50 mL)+ All buffer and sample delivery was performed by peristaltic pump+ Diluted DEAE fractions (;100 mL) were loaded onto a 2+6 ϫ 26 cm column containing heparin agarose (Sigma)+ The column was washed, then eluted with a 1800-mL gradient from 40 to 500 mM KCl in DB+ xPARN activity was assayed as above, using 5 mL of every fifth fraction+ For polypeptide identification, active heparin fractions were pooled and loaded (0+25 mL/min) directly onto a 0+5 ϫ 5 cm poly(A)-Sepharose (Pharmacia) column equilibrated with 500 mM KCl in DB on a Beckman HPLC dual-pump system with a 5-mL sample loop+ The column was washed until the cessation of baseline drift and eluted with a step to 1+5 M KCl in DB at 1 mL/min+ Eight 2-mL fractions were collected starting 4 min after start of elution+ Activity was recovered by concentrating and desalting in a Centricon C-30 ultrafiltration device (Amicon)+ Apparently homogeneous xPARN (p62) was obtained by loading desalted and concentrated activity from poly(A)-Sepharose onto a 0+5 ϫ 5 cm MonoQ column developed with a gradient to 400 mM KCl in DB over 20 min+ Silver staining of protein gels was performed as described previously (Heukeshoven & Kernick, 1985)+ As a source of partially purified xPARN, heparin agarose fractions were pooled and loaded directly onto a 0+5 ϫ 5 cm HAP (Bio-Rad ceramic hydroxyapatite) column that was eluted with a linear gradient to 150 mM KPO 4 in 20 min at 1 mL/min+ Fractions were then analyzed by TCA precipitation/SDS-PAGE or deadenylation assay or both+ For some experiments, active HAP fractions were further purified on MonoQ as described above+…”

Autocrine regulation of matrix metalloproteinase-9 gene expression and secretion by tumor necrosis factor-α (TNF-α) in NB4 leukemic cells: specific involvement of TNF receptor type 1

“…After electrophoresis gels were stained with silver according to the method of Heukeshoven and Dernick. 29 …”

High‐throughput negative detection of SDS‐PAGE separated proteins and its application for proteomics

“…Glutaraldehyde (GA)-silver nitrate staining using GA as a sensitizer was performed using a modified method described by Heukeshoven and Dernick [26]. Briefly, after electrophoresis, gels were fixed in 125 mL of 40% v/v EtOH, 10% v/v HAc solution for 30 min, and then reacted in 125 mL of 6.8% sodium acetate, 0.125% GA and 0.2% sodium thiosulfate solution for 30 min, and finally washed in 125 mL DW for 3 Â 5 min.…”

“…Although Coomassie brilliant blue stain is a reproducible, low-cost, and MS compatibility method, but it is relatively insensitive and time-consuming [2][3][4]. Silver staining is the most sensitive staining method for the visualization of protein in gels, up to 100 times more sensitive than Coomassie brilliant blue staining, but it lacks reproducibility and shows poor linearity with protein concentration [5][6][7][8][9].…”