Enhanced sensitivity of Gelcode silver staining by glutaraldehyde fixation of proteins in two‐dimensional electrophoresis

Abstract

The staining of proteins in polyacrylamide gels using Gelcodes silver staining can be enhanced approximately tenfold by fixing with glutaraldehyde. The glutaraldehyde results in the formation of a protein-aldehyde complex. This complex retards the diffusion of low molecular weight proteins from the gel and increases staining sensitivity by increasing the amount of metallic silver deposited within the protein-aldehyde complex.The application of silver as a highly sensitive method for the detection of proteins in electrophoresis [ 1, 21 is based on a modification of histological silver-staining techniques. Various modifications of this technique have recently been introduced 13-61 in an effort to detect subnanogram levels of proteins in two-dimensional polyacrylamide gel electrophoresis. Some of these methods have incorporated glutaraldehyde or formaldehyde [6-81, which form protein-aldehyde complexes that participate in the deposition of metallic silver. Current methods used in our laboratory with either ammoniacal silver or silver nitrate have resulted in poor staining ofproteins with molecular weights ofless than 20 000. Therefore, we have modified the current Gelcoden (Health Products, Inc., South Haven, Michigan) procedure [9l to include glutaraldehyde. This modification has resulted in a rapid and efficient staining procedure which is about ten times as sensitive as the current Gelcode method. This apparent tenfold increase is based upon the appearance of spots observed when gels are fixed with glutaraldehyde but that are only seen by the normal Gelcode staining method when the protein concentration is increased tenfold. E. coli proteins (M, 8000 to 30 000) were obtained by fractionation of a cell lysate over a gel filtration column (Sephadex G-50), which had been calibrated by standard techniques. The fractions were lyopholized and then resuspended at a concentration of 10 mglml in sodium dodecyl sulfate (SDS) solubilizing solution (2 % SDS, 50 mM cyclohexylamino-ethanesulfonic acid (CHES), pH 9.5, 10 % glycerol). Samples containing from 1 to 250 pg were then subjected to two-dimensional electrophoresis. The two-dimensional electrophoresis was performed according to Anderson's ISO-DALT procedure [ 10, 1 1 I. The first dimension (isoelectric focusing) was run for 10 000 Vh using the following mixture of carrier ampholytes: Pharmalytes (Pharmacia) pH 2.5-5, 20 %; pH 5-8, 40 %; pH 6.5-9, 20 % and Ampholines (LKB) p H 3.5-10, 20 %. The second dimension was performed using a 15-22 % acrylamide gradient in 25 mM TRIS, 0.2 M glycine, 35 mM. SDS buffer, which resolvedproteins ofaM,from 2700 to 113 000. M , was determined by least square fit using Gelcode and Pharmacia molecular weight markers. The gels were fixed in 50 % ethanol11 2 % acetic acid for 1 h (200 ml per 16 x 16 x 0.15 cm gel) to remove carrier ampholytes and any acrylamide monomer. The gels were then placed in 2.5 % w/v glutaraldehyde for 1.5 h for further fixing and proteins crosslinking. We have found the grade of glutaraldehyde to be critical to obta...

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

“…After boiling, the samples were placed on ice. Per sample 15 #1 was analysed on 10~o polyacrylamide gels [20] that were subsequently stained for protein with AgNO 3 according to Slisz and van Frank [34]. Immunoblotting was carried out as described by Hovenkamp-Hermelink et al [9] using antiserum raised against potato GB S S [42].…”

Accessory gland proteins of males in the male‐diphenic ant Cardiocondyla obscurior

“…After SDS-PAGE, the gels were fixed each in 200 mL of fixation solution (50% methanol, 10% acetic acid). Before silver staining overnight in accordance with the protocol of Blum et al (1987) glutaraldehyde fixation was used to enhance staining (Slisz & Van Frank, 1985). Image and statistical analysis of gels was performed with progenesis samespots software (Nonlinear Dynamics Limited, U.K.).…”

A comparison between low background silver diammine and silver nitrate protein stains

“…Early protocols using only silver nitrate (e.g. [2-41) often used oxidizing agents such as dichromate to prevent spurious silver ion reduction in the matrix, but more recent protocols frequently use reducing or sulfiding agents such as glutaraldehyde [5,6], dithiothreitol (DDT) [7,8], dithionite [9] or thiosulfate [lo]. These agents are believed to act by producing minute silver metal or silver sulfide deposits at the protein level, which act as nucleation center for silver image formation [ll].…”