Isolation of a component from commercial coomassie brilliant blue R-250 that stains rubrophilin and other proteins red on polyacrylamide gels

Commercially available Coomassie Brilliant Blue R-250 (C.I. 42660) is a popular and useful dye that stains most proteins blue on polyacrylamide gels. Some proteins from brain (rubrophilin), collagens, histones and parotid gland proteins are distinctly red when stained with Coomassie Blue. Commonly used Coomassie Brilliant Blue R-250 preparations may contain more than 30 distinct colored and fluorescent components that can be separated on silica gel chromatographic columns. A specific component has been isolated on silica gel columns that stains rubrophilin and other proline-rich proteins a reddish color. Fast atom bombardment mass spectrometry of the isolated rubrophilin staining principle indicates a molecular weight of 634 as compared to 826 for the major dye in the original Coomassie Brilliant Blue R-250. Infrared spectrometry is consistent with a difference between the rubrophilin staining principle and Coomassie Brilliant Blue R-250 of a toluene sulfonic acid residue.     

Staining properties of bovine low molecular weight hydrophobic surfactant proteins after polyacrylamide gel electrophoresis.

Reports describing polyacrylamide gel electrophoresis patterns of bovine hydrophobic surfactant proteins are not consistent. In this study, we found unusual staining characteristics of these proteins that may explain some of these inconsistencies. Low molecular weight surfactant proteins extracted from bronchoalveolar lavage with organic solvent are partially delipidated with Sephadex LH-20 chromatography using chloroform and methanol. Fractions from the first protein peak are dried under nitrogen then subjected to SDS electrophoresis on 20% polyacrylamide gels. Under nonreducing conditions, silver staining identifies 5- and 26-kDa bands, and Coomassie blue identifies 6-, 12-, and 26-kDa bands. When gels are stained with Coomassie blue then silver, the 5- and 26-kDa bands stain with silver and 6- and 12-kDa bands remain stained with Coomassie blue. If gels are first stained with silver then Coomassie blue, similar results occur. We modified the silver staining protocol by treating gels with dithiothreitol or 2-mercaptoethanol after electrophoresis. With this modification, 5-, 6-, 12-, 26-, and also 17-kDa bands are identifiable. Using the modified protocol and restaining gels previously stained with silver, 6-, 12-, and 17-kDa bands that were not identified previously all became visible. In further experiments, protein bands of 6-, 12-, and 26-kDa that were identified by Coomassie blue were electroeluted under nonreducing conditions. After electrophoresis of the eluted 26-kDa protein, bands of 17-, and 26-kDa under nonreducing, and 8-kDa only under reducing conditions, were apparent by using the modified silver protocol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Staining protein in isoelectric focusing gels with fast green

A rapid, simple technique for staining proteins in isoelectric focusing polyacrylamide gels was demonstrated using fast green in 10% acetic acid. Fast green has the distinct advantage of not binding to ampholytes, thus staining only protein. Maximum staining was achieved within 5 min, and bands were visible after 3 to 6 h of destaining. Background stain removal, however, was not complete until 72 h after placing gels in a diffusion destainer. Gel quantitation was demonstrated with actin using fast green and Coomassie brilliant blue R-250. A standard curve prepared with fast green was linear from 0.5 to 8 μg of actin in contrast to Coomassie brilliant blue R-250 which provided linearity from 0.1 to 2.5 μg actin. Application of fast green staining to quantitation of α-actin from cultured muscle satellite cells has been demonstrated.     

Advantages of picrate fixation for staining polypeptides in polyacrylamide gels

When acetic acid-urea polyacrylamide gels with or without Triton X-100 were immersed in 0.1 M Na picrate, pH 7, to which 1/4 vol Coomassie blue staining solution (0.2% in 45% methanol, 10% acetic acid, 45% water) was added, proteins stained rapidly (within a few minutes in gels without Triton and within an hour in gels with Triton) with little or no background staining. Thus protein bands could be observed in a single step with no destaining. The picrate-Coomassie blue method fixed and stained a small peptide (bradykinin, nine amino acids) that was not observed in gels stained with fast green, silver, or Coomassie blue following fixation in 50% trichloroacetic acid. The picrate-Coomassie blue method gave high-contrast bands suitable for densitometry. Gels containing sodium dodecyl sulfate were also stained by the picrate-Coomassie blue method if they were first washed briefly (1 h) in 45% methanol, 10% acetic acid, 45% water, presumably to remove the detergent. These gels also stained rapidly with almost no background.     

Direct red 81 and amido black stain proteins in polyacrylamide electrophoresis gels within 10 min

Proteins separated by SDS-polyacrylamide gel electrophoresis can be stained with organic dyes, the most popular being Coomassie brilliant blue R-250. Coomassie R-250 staining of ovalbumin in an SDS-PAGE gel increased linearly from 2.5 to 60 min. Direct red 81 and amido black staining approached saturation in 10 min. Scatchard analysis showed that the number of direct red 81 and amido black ligands bound to ovalbumin was fourfold higher than that of Coomassie R-250. Direct red 81 and amido black stain proteins in an SDS-polyacrylamide electrophoresis gel in 10 min.     

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.     

Simultaneous staining of proteins during polyacrylamide gel electrophoresis in acidic gels by countermigration of Coomassie brilliant blue R-250

A method for the simultaneous staining of proteins during polyacrylamide gel electrophoresis with Coomassie brilliant blue R-250 at pH 2.5 is described. Calf thymus whole histone and cytochrome c were stained by this method and the results obtained were similar to that obtained by staining after electrophoresis.     

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.     

A fast silver staining method for protein detection after isoelectric focusing in agarose gels

A sensitive staining method was developed for detecting proteins in agarose gels after isoelectric focusing. Its sensitivity is about 20 times that of the Coomassie blue R-250 staining technique, and the time required is only 10 min.     

Sodium dodecyl sulfate-gel electrophoresis: staining of polypeptides using heavy metal salts

Water-soluble salts of several heavy metals were examined for their ability to stain polypeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Brief gel exposure (5 min or less) to cobaltous acetate or chlorides of copper, nickel, and zinc produced negatively stained protein patterns that were qualitatively indistinguishable from those of parallel gels stained with Coomassie blue R-250. Protein patterns could be visualized less than 1 min after treatment of gels with zinc chloride; the threshold of detection was estimated at about 10-12 ng protein on standard-size slab gels. Test samples including human erythrocyte membranes, sialoglycoprotein (glycophorin) extracts, and commercial molecular weight protein standards were used to establish the scope of these stains. Protein patterns visualized by the heavy metal salts were compared and contrasted with profiles seen with three widely used silver stains. Proteins from gels treated with copper or zinc chloride could be easily recovered by simple diffusion; this makes feasible both analytical and preparative electrophoretic applications of the staining procedure. A mechanism is proposed to explain the observed protein staining by heavy metal salts.     

A method for staining and stabilizing peroxidase activity in polyacrylamide gel electrophoresis

A procedure was developed for a rapid double staining of peroxidase and other proteins in the same polyacrylamide gels using guaiacol and Coomassie blue. The distinguishable colored bands of peroxidase isozymes and proteins are stable for at least 8 months.     

Advantages and disadvantages of silver staining of proteins in polyacrylamide gel

Sensitivity of protein staining with Serva blue G-250 (Coomassie brilliant blue G-250 analogue) in polyacrylamide gel was determined. It has been shown that protein staining with 0.1% Serva blue G-250 results in the recovery of 80 to 35 ng of single protein, that is almost 10 times higher than reported previously for Coomassie brill. blue G-250 (or R-250) staining. The comparison of the sensitivity of Serva blue G-250 protein staining in PAAG and AgNO3 has shown that AgNO3 staining was approximately 18-30 (but not 100 times, as it had been thought before) times more effective for the majority of proteins under study. Silver staining of some proteins, for instance ribonuclease and a number of retrovirus-specific structural proteins, was of lower efficacy. Thus, to obtain reliable results protein electrophoresis in PAAG should be followed by both staining procedures.     

Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins.

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.     

Hydrazinoacridine staining of proteins and glycoproteins in polyacrylamide gels

The histochemical method for staining polyaldehydes in tissue sections with p-hydrazinoacridine has been adapted for use in polyacrylamide gels. While staining of histological preparations was reported to be specific for polyaldehydes and independent of bisulfite, both glycoproteins (β chain of haptoglobin) and nonglycoproteins (lysozyme and α chain of haptoglobin) were stained following periodate oxidation, and satisfactory results were highly dependent on the presence of bisulfite. Hydrazinoacridine staining of periodate-treated gels produced an extremely sensitive fluorescent labeling of the haptoglobin β chain and also stained haptoglobin α chain and lysozyme. The proteins could be visualized under visible light as yellow bands which were scanned spectrophotometrically at 440 nm. The β chain of haptoglobin could be subjectively distinguished from the nonglycoproteins both by differential intensity of staining with hydrazinoacridine and Coomassie brilliant blue and by the yellow nature of the fluorescence. The sensitivity of hydrazinoacridine staining of the β chain of haptoglobin compared favorably to that of the commonly used periodic acid-Schiff staining procedures and provided the advantage that nonglycoproteins in complex mixtures could be localized in the gels.     

An improved staining technique for precipitin bands in agar or agarose gels

A method for the staining of proteins in agar and agarose gels using three stains simultaneously and a mordant is described. When compared with conventional Coomassie brilliant blue R-250 staining procedures, it requires a comparable time expenditure but has the following advantages: 1) it is threefold to fourfold more sensitive; 2) there is increased photographic resolution on conventional photographic material; and 3) the stain has a long shelf-life and does not fade under normal lighting conditions. Conditions for the washing and drying of gels are discussed.     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

Extraction of fixed, stained protein bands from gels for micro amino acids analysis using o-phthaldialdehyde.

A method is presented for extraction of fixed, stained protein bands from polyacrylamide gels suitable for automated fluorescence analysis of amino acids using o-phthaldialdehyde. Bands, containing microgram quantities of protein and stained with Coomassie blue, are extracted from homogenized gel slices with sodium dodecyl sulfate. The Coomassie blue and sodium dodecyl sulfate do not interfere with the amino acid determination, and contamination by ammonia from the gels is low. The method has been applied to the analysis of human carbonic anhydrase C, and the amino acid composition is found to be similar to that obtained by other methods requiring larger amounts of protein.     

The use of N,N,N',N'-tetramethylphenylenediamine to detect peroxidase activity on polyacrylamide electrophoresis gels

N,N,N',N'-Tetramethylphenylenediamine (TMPD) acts as an effective indicator of peroxidase activity on polyacrylamide electrophoresis gels. The test is easy to perform, rapid, sensitive, and reliable. The procedure produces vivid bright blue bands (Wursters blue) on a clear background. TMPD and Wursters blue did not interfere with a number of other electrophoresis stains subsequently applied. These included total protein staining with Coomassie blue, and a number of pigment producing electrophoresis stains used to investigate melanogenesis-related enzymes in the black yeast Phaeococcomyces sp.     

Reverse sister chromatid differential staining by Coomassie Brilliant Blue R-250

A sister chromatid differential staining pattern is observed if chromosomes replicate for two cycles in the presence of 5-bromodeoxyuridine (BUdR) and are subsequently stained in Hoechst 33258, irradiated with black light, and then stained in Coomassie Brilliant Blue R-250. In this pattern the chromatids containing DNA that is bifilarly substituted with BrdUrd are darkly stained and the chromatids with DNA that is unifilarly substituted are lightly stained. This staining pattern is the reverse of that found when slides are stained in Hoechst plus Giemsa. Slides stained with either Giemsa or Coomassie Blue can be destained and restained repeatedly with the other stain to alternate the pattern observed.     

Specific aspects of detection of peroxidase isozymes and their genetic control in barley

Identical specimens were separated by electrophoresis in two gels to detect and fix peroxidase isozymes. Both gels were stained by Coomassie brilliant blue for detecting proteins. One gel was previously incubated for detecting peroxidase activity. The differences in electrophoretic patterns between the gels indicate the zones of peroxidase activity. It has been shown that locus Prx 6H, controlling a low-mobility grain peroxidase (PRX 6H), is localized to barley chromosome 6. Two loci, Alb 4H and Alb 7H, controlling the biosyntheses of water-soluble proteins of barley endosperm, were localized to chromosomes 4 and 7. It has been demonstrated that barley species is polymorphic at multiple molecular forms of peroxidase.