The basis for colored silver-protein complex formation in stained polyacrylamide gels

Using a modified silver stain of Merril et al. [(1981) Science 211, 1437-1438] for staining polypeptides in sodium dodecyl sulfate-polyacrylamide gels, protein bands reproducibly stain different shades of blue, yellow, red, and gray. The procedure is highly temperature dependent, with optimal color formation at 42 degrees C. The procedure may be completed within 2 h. Color formation is due to silver ion complexes with charged amino acid side chains. The color of the silver-protein complex can be predicted if the amino acid sequence is known, although some exceptions are discussed. This provides another dimension to the characterization of proteins by gel electrophoresis.     

Amino acid analysis with fluorescamine of stained protein bands from polyacrylamide gels

Bands in stained polyacrylamide gels, containing microgram quantities of protein, have been subjected to hydrolysis and subsequent amino acid analysis with fluorescamine. Analyses on these protein bands agree with deterinations performed on aqueous solutions of the protein and with published results.     

Quantitation of stained proteins in SDS polyacrylamide gels with lysozyme as internal standard.

A simple method for the quantitation of proteins on SDS-polyacrylamide gels, with lysozyme as an internal standard, has been designed. Gels containing known weight ratios of standard proteins to lysozyme were electrophoresed, stained with Coomassie blue R250, and scanned at 550 nm. Peak areas corresponding to individual proteins were determined and the area ratios of proteins to lysozyme were calculated. Plots of area ratio vs weight ratio were linear over a limited range and were reproducible from gel to gel and thus suffice as a standard curve. We have used this method to determine accurately and precisely the amount of rhodopsin in the photoreceptor membranes of rat retinas.     

Simple and inexpensive preparation of dried polyacrylamide gels for densitometry and/or storage

Polyacrylamide gels soaked in glycerol, dried at room temperature, and sealed with a clear sheet of inexpensive polypropylene are clear, flat, flexible, and stable. The technique is simpler and less expensive than others that have been described and does not involve the use of protein glues.     

The quantitation of rat serum esterases by densitometry of acrylamide gels stained for enzyme activity.

Normal rat sera were electrophoresed on polyacrylamide gels. The gels were then stained for (a) esterase activity and (b) protein content. Subsequent analysis on a densitometer permitted quantitative estimation of both enzyme activity and protein concentration. (The specific activity (per mg of protein) of individual serum esterases were then calculated.) Two previously unreported esterases (Bands A and D), were identified and partially characterized. When carefully prepared, non-activated plasma was used, no differences in enzyme activity were found compared to serum.     

Electron densitometry of stained virus particles

Methods are described for determining the relative mass of particles in electron microscope specimens through the measurement of photographic densities in recorded images. These methods were applied to a quantitative study of the amounts of electron stains that could be associated with the particles of tomato bushy stunt virus (BSV) and tobacco mosaic virus (TMV). In the pH range above 2 where the viruses are stable, the amount of stain absorbed is too small to produce adequate contrast in the electron microscope. Maximum stain absorption was achieved at pH about 1 where with several reagents and combinations of reagents the mass of BSV could be increased to about four times that of the unstained particles. Optimum results were obtained with phosphotungstic acid alone or in combination with Pt, Th, or La ions. Since the pH conditions for high stain absorption are normally destructive, morphology is satisfactorily preserved only when the phosphotungstic acid is applied in concentrations of 10 per cent or greater or when the use of destructive reagents is preceded by a preliminary fixation under mild conditions. Maximum staining of TMV increased the mass of the particles to about two times that of the unstained. Estimates of the mass of heavily stained BSV particles indicate that their density is 3.3 gm./cm.(3) The high internal hydration of BSV probably accounts for the greater stain absorption and penetration compared to those of TMV which has very low or zero internal hydration. Anomalous images resulting from the use of electron stains are shown and discussed.     

Transmission densitometry of stained nitrocellulose paper

We report a simple solution to the problem of quantitative densitometry of stained nitrocellulose paper. By immersing the paper in a household lubricating oil of matching refractive index, the light-scattering properties of the paper are largely eliminated, allowing precise transmission densitometry in any flat bed densitometer. The method was evaluated on immunochemically stained Western blots of the proteinases cathepsins B and L. An approximately linear relationship was found between the integrated absorbance of the stained zone and the logarithm of the amount of protease applied to the polyacrylamide gel over the range of 150 to 350 ng of cathepsin B and 50 to 250 ng of cathepsin L.     

Polymerase chain reaction amplification products separated on rehydratable polyacrylamide gels and stained with silver

Separation of polymerase chain reaction (PCR) amplification of specific fragment length polymorphisms was carried out on rehydratable polyacrylamide gels on a horizontal flat slab system. A discontinuous sulfate-borate buffer system was employed on 5-8% T gels crosslinked with 3.5% C. Samples were diluted in leading sulfate ion buffer at 1/10 the ionic strength of the separating gel buffer and placed directly onto the surface of the rehydrated gels in 0.5-10 microliters volumes. The trailing ion and counterion were contained in a gel plug and placed directly onto the anodal and cathodal ends of the gel, and the electrodes placed directly onto the surface of the gel plugs. Filter paper wicks, soaked in diluted leading ion buffer, were placed along each side to lower the ionic strength of the edges, thereby increasing mobility at the edge and thus preventing smile effects. The gel-gel contact of the plug and separating gel prevent the production of a junction potential which occurs between dissimilar materials such as a paper wick and the gel. Ten- to 20-cm separations were carried out from 2-5 h, respectively, and resolution in the 20 cm system was 1.6-4 bp (base pairs) between 100 and 500 bp, 4-7 bp between 500 and 1000 bp, 12-20 bp between 1000 and 2000 bp and about 50 bp between 2000 and 3000 bp. Between 3000 and 4000 bp, resolution fell off to +/- 100 bp. Sensitivity, using a silver stain, indicated that one could readily distinguish less than 10 pg of DNA per mm width on the gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metachromasy of peripheral nerve collagen on polyacrylamide gels stained with Coomassie brilliant blue R-250.

Endoneurial collagen stains metachromatically with Coomassie brilliant blue R-250 (C.I. 42660) when peripheral nerve proteins are solubilized with urea and SDS and then subjected to SDS-polyacrylamide gel electrophoresis. The metachromasy is reproducible under different fixing and staining conditions, but was exhibited only by Coomassie blue R-250 of the four triphenylmethane dyes tested. A method is presented for measurement of the degree of metachromasy on SDS gels and the detection of collagen in homogenates of whole tissue.     

Detection by the use of silver ions of additional protein zones in gradient polyacrylamide gels stained with Coomassie

A simple method of revealing the additional zones of proteins in gradient polyacrylamide gels, preliminary dyed Coomassie by means of silver ions is described. The dyeing of Coomassie allows to avoid the time-consuming stages of preliminary treatment of gels as well to reveal more sensitive zones in gels. On the second stage of dyeing silver minor zones appear there which were not seen while Coomassie was dyed. The suggested method preserves high sensitivity characteristic of the methods of gel dyeing with silver.     

High sensitivity method for fluorofore detection in gradient polyacrylamide slab gels through excitation by laser light: application to glycoproteins stained with concanavalin A-fluorescein isothiocyanate

An easy-to-assemble apparatus for the laser-light excitation of fluorofores in polyacrylamide gels is described. The assemblage is made up of a continuous-wave ion-argon laser with adjustable power output, a beam diffuser, appropriate filters to block excitation light, and a photographic camera. With this setup a minimum 20-fold increase of sensitivity was obtained for fluorofore detection in polyacrylamide gels as compared to the more conventional uv-light excitation using a commercial preparation of Con A-FITC (concanavalin A-fluorescein isothiocyanate) as reference molecule in the gel. The same apparatus, used to analyze the Con A-positive glycoproteins contained in serum Cohn fraction IV separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed a number of fluorescent components in a wide range of relative intensities while uv-light excitation showed none. Acrylamide concentration in the gel is critical, since a working limit of between 10 and 12% has been found, above which the diffusion of Con A-FITC in the gel, necessary to label glycoprotein bands, is hampered. The system described here also permits the optimization of detection of minor components not otherwise observable by conventional light excitation, because light power, angle of incidence, and beam divergence can be adapted to analyze specific areas of the sample gel.     

Protein transfer from fixed, stained, and dried polyacrylamide gels and immunoblot with protein A-gold

The method of electrophoretically transferring proteins from fixed and stained polyacrylamide gels onto nitrocellulose paper has been reevaluated. It is shown that the tedious destaining of gels is not necessary because Coomassie brilliant blue, although it binds tenaciously to nitrocellulose paper, does not reduce the transfer efficiency of proteins. However, its presence impairs the visibility of proteins as detected, for instance, by the immunogold technique. Therefore, a rapid method for the complete removal of the stain from the nitrocellulose paper after completion of the immunogold procedure was developed. Furthermore, it is shown that proteins from dried polyacrylamide gels can still be transferred onto nitrocellulose sheets with an efficiency of approximately 50% compared to proteins transferred from fixed gels.