A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.     

A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels

We have simplified the highly sensitive silver stain of R. C. Switzer III, C. R. Merril, and S. Shifrin (1979, Anal. Biochem.98, 231–237) for visualizing proteins in polyacrylamide gels. We have reduced the number of steps in the procedure from 10 to 6, simplified the reagents in each step, and reduced the amount of silver required by a factor of 10, thus greatly reducing the expense of the procedure. In common with the original silver stain, our procedure is 100 times more sensitive than Coomassie brilliant blue and is comparable in sensitivity to radioautography of radioactively labeled proteins.     

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

Silver stain for proteins in ultrathin polyacrylamide gels: a sensitive precipitation technique

A rapid and highly sensitive silver stain for visualization of proteins on ultrathin isoelectric focusing gels is described. This procedure is based on the specific interaction of silver and bromide ions in the presence of proteins and appears to involve a precipitation reaction. The technique requires only two reaction solutions, a silver nitrate and a potassium bromide solution. Silver consumption is very low because the silver nitrate solution is reusable. This procedure is well established for proteins separated by isoelectrofocusing in ultrathin gels.     

Ability ofVibrio vulnificus to obtain iron from transferrin and other iron-binding proteins

The ability of virulent and avirulent strains ofVibrio vulnificus to overcome iron limitations by using iron bound to iron-binding proteins was examined. While no strains were able to obtain iron from lactoferrin or ferritin when these proteins were not fully saturated with iron, growth was enhanced by the iron-saturated form of these proteins. None of the strains was able to scavange iron from 30% saturated transferrin, but there were strain differences in the ability to obtain iron from the saturated form. The virulent strains were able to compete more efficiently with transferrin when it was fully saturated with iron than were the avirulent strains.     

A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels

A sensitive silver stain for detecting bacterial lipopolysaccharides in polyacrylamide gels is developed by modifying the silver-staining method used for proteins (cf. R. C. Switzer III, C. R. Merril, and S. Shifrin, Anal. Biochem.98, 231–237 (1979). Lipopolysaccharides are analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by visualization with either the modified silver stain or periodic acid-Schiff stain. The lipopolysaccharides are stained dark brown by the silver stain. The silver stain is 500 times more sensitive than the periodic acid-Schiff stain and can detect less than 5 ng of rough type lipopolysaccharides. Analyses of 5μg of smooth-type lipopolysaccharides from Salmonella typhimurium and Escherichia coli O111: B4 show each to have 30–40 components of different molecular weights. The use of a lipopolysaccharide having a known structure and variable numbers of repeating units in the O side chain, such as one of the two lipopolysaccharides mentioned above, as molecular weight markers is proposed for the estimation of the molecular weights of other lipopolysaccharides or their components. The lipopolysaccharides can also be stained grayish green, but become grayish blue with a heavy sample load, using a silver-based color-staining method (D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis2, 135–141 (1981)).     

A rapid sensitive silver stain for polypeptides in polyacrylamide gels

The use of silver to detect polypeptides was originally achieved by modifying tissue stains. By adapting methods of photochemistry we have developed a new silver stain for polypeptides which is nearly as sensitive but much more efficient than these earlier procedures. The new silver stain utilizes only three solutions and allows protein patterns to be visualized within 50 min. Its sensitivity is 100 times that of the Coomassie blue stain.     

A sensitive urea-silver stain method for detecting trace quantities of separated proteins in polyacrylamide gels

An ultrasensitive method using a urea-silver staining procedure to detect trace quantities of proteins in polyacrylamide gels (PAGE) is described. This technique is sensitive enough to detect picogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels. The major advantages of our method are that it provides a clear background and it is more sensitive than other techniques allowing it to substitute for radioisotopic techniques in some cases.     

Detection of proteins and sialoglycoproteins in polyacrylamide gels using eosin Y stain

An eosin Y staining technique that permits detection of various proteins, including membrane sialoglycoproteins, in polyacrylamide gels is described. The sensitivity of the eosin Y staining method is comparable to silver staining. In addition, there is an added advantage of the antigen icily of the stained proteins being retained in a Western blot. Details of the procedure to obtain optimal staining results are described.     

Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity

Filter-paper disks of uniform size were chemically modified by the introduction of isonitrile functional groups. Avidin was then covalently linked to the disks in a four-component condensation reaction involving disk isonitrile groups and avidin carboxyl groups in the presence of a water-soluble aldehyde and an amine. Quantitative assay of unknown d-biotin solutions could be conveniently carried out with such avidin-cellulose disks by a two-step procedure: (i) immersion in the unknown sample, and (ii) exposure to an excess of radioactive biotin. Based on the known total capacity of the disks for biotin, the amount of unlabeled biotin extracted from solution by avidin-cellulose disks could be easily estimated.     

A faithful double stain of proteins in the polyacrylamide gels with Coomassie blue and silver

The conditions for prior fixing of proteins in a gel in order to attain a greater degree of faithful silver staining and sensitivity were examined. Fixing with formaldehyde enhanced the retention of proteins in a gel, particularly basic proteins such as histones and ribosomal proteins. The gel, one stained with Coomassie blue and following the removal of the free dyes, is capable of undergoing silver staining, and, moreover, the prestain considerably enhanced the staining intensity of various proteins differing in basicity in subsequent silver staining. Coupling the formaldehyde fixation with Coomassie brilliant blue prestain afforded a reproducible and pronounced stainability of various proteins.     

A specific stain for sulfhydryl groups in proteins after separation by polyacrylamide gel electrophoresis

A new method is described for specifically staining protein sulfhydryl groups after the proteins have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in slab gels. The stain will detect as little as 0.25 microgram of lysozyme and 1 microgram of most other proteins; the range of sensitivity for a specific protein depending on its sulfhydryl content. Proteins with no cysteine residues (type I collagen) and glycoproteins do not cause spurious staining.     

A sensitive silver stain for proteins in agarose gels

A silver stain for proteins in agarose gels which is at least 10 times as sensitive as Coomassie blue is described. The method is simple to use and is particularly useful for the study of protein bands in the gamma region on electrophoresis of fluids such as cerebrospinal fluid in which the protein concentration is low. It readily detects bands of IgG containing 20 to 40 ng/band (approx 3 to 6 ng of IgG/mm2 of gel).     

A specific stain for α-glucosidases in isoelectric focusing gels

The separation of the four diastereomers of β,γ-bidentate Cr · ATP using reverse-phase HPLC techniques is described. This technique provides complete resolution of the diastereomers within 10 min and relies on the use of methanesulfonic acid (in the ionized form) as an ion-pairing agent. To identify the screw sense of these resolved isomers, the CD spectra of each isomer were done, and substrate and inhibition specificities were examined using hexokinase. The results were then correlated with the isomeric assignments made by D. Dunaway-Mariano and W. W. Cleland (1980, Biochemistry19, 1506–1515). Further studies included the monitoring of isomer interconversion at pH 6.2 to an equilibrium concentration of all four, and specific rotation measurements of the pure isomers at pH 2.5, 23°C, and 546 nm.     

59Fe]Ferrous bathophenanthroline sulfonate: a radioactive stain for labeling proteins in situ in polyacrylamide gels

The use of a protein stain, [⁵⁹Fe]ferrous bathophenanthroline, to radioactively label proteins in polyacrylamide gels after electrophoresis using simple staining and destaining procedures is described.     

Detection of nitrogenase components and other nonheme iron proteins in polyacrylamide gels

The two Fe-containing component proteins of nitrogenase in crude cell-free extracts of N₂-fixing bacteria can be detected as pink bands upon treatment of polyacrylamide gel with α, α-dipyridyl and mercaptoacetic acid.This technique is useful in studies of mutant strains lacking N₂-fixing activity. It might be of value in the analysis of other nonheme iron proteins.     

Aerobic ferrisiderophore reductase assay and activity stain for native polyacrylamide gels

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.