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.     

Fast and efficient method for detection and estimation of proteins

A quick, simple, inexpensive and sensitive method is described to stain and quantitate proteins on nitrocellulose papers. The proteins may be spotted or transferred from polyacrylamide gels by Western blotting. The procedure involves non-radioactive iodination of the polypeptides by chloramine T and potassium iodide followed by detection of bound iodine with starch. The method is more sensitive and much quicker than Coomassie brilliant blue staining and may be used for quantitation or detection of proteins in unknown samples. Another major advantage of this procedure is that ionic or nonionic detergents, although at higher concentrations causing the sample to disperse more broadly in the membranes, do not affect the staining procedure. Further, this method may be used for detection of proteins bound to papers that have high affinity for proteins such as the Zeta probe membranes.     

Studies and critique of Amido Black 10B, Coomassie Blue R, and Fast Green FCF as stains for proteins after polyacrylamide gel electrophoresis.

The properties of amido black 10B (C.I. 20470), Coomassie blue R (C.I. 42660), and fast green FCF (C.I. 42053) as protein stains, along with a few comments on Coomassie blue G (C.I. 42655), are presented and dye impurities and their effects on protein-dye binding within gels are discussed. All three dyes produced metachromatic effects with some proteins. Problems encountered with long-term stability and fixation of certain maize seed proteins are reported along with procedures for overcoming them. The low solubility of Coomassie blue R in trichloroacetic acid prevented maximum staining and destaining within a reasonable time, whereas other solvents allowed diffusion of some proteins during staining. Coomassie blue R binds to proteins in much higher amounts than do amido black and fast green, which accounts for its sensitivity in detection of protein bands in gels. Procedures for obtaining maximum contrast with photographs are also outlined.     

Chloramine T-mediated radioiodination of proteins on intact polyacrylamide slab gels

A simple, inexpensive method is described here for the radioiodination of proteins on intact, developed polyacrylamide gels. The method is based on the chloramine T iodination procedure which yields proteins containing ¹²⁵I label specifically in tyrosine residues. When employed with intact polyacrylamide gels, our method allows detection of proteins in amounts too small to be observed by chemical stains, including Coomassie blue. The procedure should, therefore, be useful for analyzing protein mixtures where only a small amount of material is available or for assaying trace contaminants in purified protein preparations. Proteins radioactively labeled by our method are suitable for further analysis by proteolytic cleavage followed by peptide mapping or “fingerprinting.”     

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.     

Staining and destaining polyacrylamide gels: A comparison of Coomassie blue and fast green protein dyes

A method using fast green dye for quantitation of immune serum globulin and its products of fragmentation in polyacrylamide gels has been developed. Although fast green is shown to be somewhat less sensitive than the usually used Coomassie blue stain, the former dye does not suffer from selective loss of dye due to temperature or alcohol content of the destaining solution. Destaining of fast green-stained gels is accomplished rapidly and efficiently by the described destaining procedure without an accompanying loss in quantitation.     

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.     

Direct detection of antigens in sodium dodecyl sulfate-polyacrylamide gels

We describe a simple immunochemical technique for the detection of specific antigens by antibody binding in polyacrylamide gels. Proteins are solubilized in sodium dodecyl sulfate and separated by electrophoresis in SDS-slab gels. Following fixation and removal of SDS, gel strips are incubated with normal or immune sera. After washing out unbound antibody, the gel strips are either fixed and stained with Coomassie blue or exposed to anti-immunoglobulin conjugated to horseradish peroxidase. The region(s) of antibody-antigen binding are determined from densitometric scans of the Coomassie blue-stained gels versus controls or by treatment of the gels with diaminobenzadine to localize the peroxidase. We have used this technique successfully with antibodies against fibroblast myosin, bovine serum albumin, goat immunogolbulin, the 220,000-dalton fibroblast cell-surface protein, and chicken gizzard filamin. Lectin-binding proteins can also be detected by substituting lectins for the immunoglobulins.     

Selective silver-staining methods for RNA and proteins in the same polyacrylamide gels

Two silver-staining methods for selective and ultrasensitive detection of RNAs and proteins in the same polyacrylamide gels were developed, both derived from procedures recommended for protein staining. The first, a double-staining technic with Coomassie brilliant blue and ammoniacal silver, allows visualization of RNAs as negative bands and proteins as dark brown bands. The second is also a double-staining technique, but uses silver in both steps. This second method develops the RNA bands first and then the protein bands. These techniques, especially the second, permit characterization of the different components of ribonucleoproteic complexes in the same electrophoresis gels.     

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)     

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.     

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.     

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.     

Quantitation of proteins by elution of Coomassie brilliant blue R from stained bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A simple method for the extraction of Coomassie brilliant blue R from stained protein bands excised from polyacrylamide gels is described. Spectrophotometric measurement of the eluted dye forms the basis of a sensitive assay to quantitate proteins in gels in the range 0.5-10 micrograms. The method requires no unusual equipment and is suitable for measurement of multiple samples. The polypeptide is not extracted and remains available for further analysis. The technique has been applied to three proteins and gels of various acrylamide percentages.     

Silver stain for proteins on a cellulose acetate membrane

A rapid and sensitive silver staining method to detect proteins on a cellulose acetate membrane has been established. This method is achieved by modification of the silver-based color staining for detection of proteins in polyacrylamide gels [D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis 2, 135-141 (1981)] and applied to our new type of two-dimensional electrophoresis for analysis of proteins on a cellulose acetate sheet [T. Toda, T. Fujita, and M. Ohashi, Anal. Biochem. 119, 167-176 (1982)]. Maximal sensitivity of silver stain for proteins on a cellulose acetate membrane can be obtained by an optimal balance between deposition of silver on the protein and on the background. Certain kinds of proteins are colored red, orange, or grayish-blue. The silver stain is 20-80 times more sensitive than Coomassie blue and some spots are visualized reproducibly by silver only. Densitometric evaluation of standard proteins stained with silver and Coomassie blue is also demonstrated. The method takes only 50 min to perform and is sensitive, simple, and reproducible.     

Visible fluorescent detection of proteins in polyacrylamide gels without staining

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.     

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

Quantitation of proteins bound to polyvinylidene difluoride membranes by elution of coomassie brilliant blue R-250

A rapid and simple method for the quantitation of stained proteins bound to polyvinylidene difluoride (PVDF) membranes via the elution of Coomassie brilliant blue R-250 is described. A mixture of standard proteins was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Spectrophotometric analysis of dye eluted from protein bands in the range of 0.5-10 micrograms gave a linear change in the absorbance at 595 nm. Maximal absorbance readings were attained following 5 min of dye elution, and the readings remained unchanged for elution times up to 60 min. The method requires no unusual reagents or equipment, is suitable for the analysis of multiple samples, and does not consume the protein in the process of quantitation. This technique provides a useful means for the quantitation of proteins bound to PVDF membranes prior to amino acid sequence determination, immunological analysis, or other biochemical characterizations.     

Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel

A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.     

Quantitation of specific proteins in polyacrylamide gels by the elution of fast green FCF

The quantitation of proteins in polyacrylamide gels stained with Fast Green FCF has been investigated using a modification of the elution technique originally described by Fenner et al. (Fenner, C., Traut, R.R., Mason, D.T. and Wikman-Coffelt, J. (1975) Anal. Biochem. 63, 595–602) for Coomassie Blue and adapted by Medugorac (Medugorac, I. (1979) Basic Res. Cardiol. 74, 406–416) for use with proteins stained with Fast Green FCF. The elution of dye from stained protein was accomplished using 1.0 M NaOH instead of aquoeus pyridine as required by the original method. The primary advantages of our modification are that the time required for protein quantitation has been considerably reduced and the use of toxic organic solvents has been eliminated. We have investigated the applicability of the method to several different proteins and our results indicate: (a) The quantity of Fast Green eluted from specific proteins is proportional to the quantity of protein applied to the gel, but varies for each individual protein. (b) The method allows quantitation over a very wide range of protein (1–800 μg). (c) Quantitation of protein is independent of the width of the stained bands as well as acrylamide concentration. (d) The method is applicable to gels of many types including disc, slab and continuous gradient gel, (e) Protein can be estimated from the patterns obtained by two-dimensional polyacrylamide gel electrophoresis. (f) The presence of Triton X-100 in gel and protein sample does not affect quantitation; the method is applicable to gels containing SDS provided that SDS is removed prior to staining. (g) Precipitation of protein with 12.5% TCA following electrophoresis does not interfere with quantitation. (h) The reproducibility of the technique is excellent, with standard deviations being less than 10% of the mean in all cases. This method appears highly versatile but requires appropriate standards for the quantitation of individual proteins.