Lipopolysaccharide interferes with the staining of lipoprotein on polyacrylamide gels

After electrophoresis of total membrane preparations of Escherichia coli B on sodium dodecyl sulfate polyacrylamide gels, and subsequent staining with Coomassie Brilliant blue, a band corresponding to the Braun lipoprotein fails to appear. This is in contrasr to similar preparations of E. coli K-12 which do display the lipoprotein uponm staining. Experiments described below indicate that failure to observe this protein in E. coli B is due to interference in the staining reaction by the lipopolysaccharide present in the membrane preparations.     

Activity staining of endoglucanases in polyacrylamide gels.

The endoglucanases of Penicillium funiculosum were analyzed for the presence of multiple forms using a modified version of the Congo red method. Postelectrophoretic slab gels were directly incubated in a solution of carboxymethylcellulose for a period as short as 15 min and then the activities were visualized by staining with Congo red. Ten distinct bands of clearances were obtained indicating the presence of at least as many multiple forms.     

Silver staining DNA in polyacrylamide gels

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.     

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.     

The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin A-peroxidase.

The plant lectin concanavalin A (Con A) possesses a remarkably specific capacity to bind primarily α-d-mannose or α-d-glucose sugar residues on macromolecules (cf. 1). The multivalent Con A will bind to carbohydrates on cell surfaces, and free binding sites on the attached Con A will bind to horseradish peroxidase which is a glycoprotein (2). Since peroxidase may be visualized by reaction with diaminobenzidine (3), it has been possible using this method to specifically “stain” carbohydrate residues on cell surface macromolecules (2, 4). The same principles for staining cell surfaces should apply to “staining” glycoproteins separated by polyacrylamide electrophoresis. In this paper, we examine the staining of glycoproteins in sciatic nerve by a Con A-peroxidase labeling technique. The method is more sensitive for mannose or glucose containing glycoproteins than the periodic acid-Schiff's (PAS) method commonly used.     

Silver staining of proteins in polyacrylamide gels

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. It is compatible with downstream processing, such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.     

Silver staining of proteins in polyacrylamide gels

An automatic method for the protein assay using Coomassie Brilliant Blue G-250 was developed and applied to the assay of urinary proteins. In developing the automatic system, the adhesion of protein-bound dye to the walls of the flow cell and tubes was found to be the most troublesome problem, by which the baseline was shifted upwardly to give positive errors. For the purpose of preventing such adhesion, the concentration of CBB was reduced to half of that used in the manual method, glass tubes and glass coils were changed to those made of Kel-F material, and the flow cell was coated with fluorine resin. As a result, the staining with protein-bound dye was nearly completely eliminated. The final system showed satisfactory ability in performance, namely, the value of a coefficient variation for the reproducibility within run was 1.3%, that for the carry over was 0–1.1%, and the recovery was 98.8%. The calibration curve was linear in a range of 0–1000 μg/ml, and 80 samples could be processed in 1 h. Thus, the present method may serve as an efficient automatic protein analyzer for routine clinical tests of urine samples.     

Fast and sensitive silver staining of DNA in polyacrylamide gels.

The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).     

Silver staining for carboxymethylated metallothioneins in polyacrylamide gels

A sensitive method for detecting metallothioneins (MTs) by use of silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs was developed. Carboxymethylation of metallothioneins is indispensable because it prevents their aggregation, thereby allowing each of them to be detected as a single band by SDS-PAGE. However, when the gel was subjected to the silver-staining method of C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert [(1981) Science 211, 1437-1438], the image of carboxymethylated purified MTs was totally negative. Pretreatment of the gel with 1% sodium thiosulfate just prior to the silver-staining procedure successfully reversed the negative image of carboxymethylated MTs. Further, they could be detected with a limit of nanogram levels per lane. This method can be applied to MTs in cell extracts from cultured cell lines treated with cadmium or to those from liver of cadmium-intoxicated mice.     

A new staining method for the assay of proteins on polyacrylamide gels

A simple method of staining proteins on polyacrylamide gel supports with a derivative of Remazol Brilliant Blue R is described. The stain is sensitive to the extent of picking up 0.5 μg of some proteins and the method is semiquantitative. Deficiencies in application and measuring techniques leading to deviations from linearity between the absorbance of the stained protein and the amount of protein are discussed.     

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.     

Selective silver staining of urease activity in polyacrylamide gels

A selective method for staining urease activity bands in nondenaturing polyacrylamide gels is described. It is based on the deposition of silver at the urease bands after incubation of gels in the presence of urea and photographic developers. Its highly sensitivity (up to 0.015 enzyme units, corresponding to 5 ng of purified urease) is based on both the silver deposition enhancement methodology and the developers used. The selectivity of the procedure is based on the local pH increase catalytically produced by the enzyme in the presence of urea. The densitometric scan of the enzyme bands gives a linear response at least in the range 0.015-0.300 urease units. This selective staining method is about 2.5 times more sensitive than the standard silver staining of proteins, in terms of detectable urease amount.     

Double staining to increase the sensitivity of protein detection in polyacrylamide gels.

As more and more researchers are examining proteins that are available only in extremely limited quantities, i.e., cellular extracts or genetic engineering products, it is critical to utilize staining methods that maximize sensitivity. The protocol we describe here--double staining of polyacrylamide electrophoresis gels with Pro-Blue (colloidal blue stain) followed by silver staining--yields an extremely sensitive, nonspecific protein stain. On average, this double-staining technique resulted in a 40-fold increase in sensitivity and intensity vs. silver stain alone. This is a tremendous return for a small investment in additional time and materials.     

Detecting proteins containing 3,4-dihydroxyphenylalanine by silver staining of polyacrylamide gels.

Proteins in which some or all of the tyrosine side chains are post-translationally modified to dihydroxyphenylalanine have been found in several invertebrate phyla. In this paper we describe the unusual silver-staining properties of these 3,4-dihydroxyphenylalanine (Dopa)-proteins in silver-stained polyacrylamide gels. Our evidence suggests that the rapid silver staining of these proteins is due to the 3,4-dihydroxyphenol ring which is a highly effective reducing agent in the alkaline development conditions used in the final step of most silver-staining procedures. Normal proteins comprising the standard 20 amino acids and tyrosine on its own, do not reduce silver under these conditions. Pretreatment of the gels with acid-dichromate solutions abrogates the rapid staining of the Dopa-proteins. This rapid silver-staining technique will facilitate the rapid screening of many additional organisms for Dopa-proteins using sodium dodecyl sulfate gels and small amounts of tissue.     

Specific staining of myo-inositol-1-phosphatase on polyacrylamide gels after electrophoresis

A sensitive staining method was developed to localise the activity of myo-inositol-1-phosphatase on Polyacrylamide gels after electrophoresis. The method can also be used for non-specific phosphatases as well as for those specific phosphatases acting upon inositol polyphosphates which are prime cellular second messengers. One or two nmol of phosphate is sufficient and less than 3 μg of purified protein will facilitate the localisation of phosphatase. If more phosphatases are present in the enzyme preparation, a combination of inhibitors can be used to suppress the activities of unwanted phosphatases and the use of specific substrates will facilitate the localisation of enzyme of interest. For nomenclature of myo-inositol phosphates recent recommendations are followed. ReferBiochem. J.,258, 1–2 (1989) andEur. J. Biochem.,180, 485–486 (1989). L-myo-inositol-1-phosphate is presently otherwise called as D-myo-inositol-3-phosphate. Dedicated to Dr. F. Eisenberg Jr., on his 70th birthday.     

Silver staining of high molecular weight proteins on large-pore polyacrylamide gels

A large-pore gel for electrophoresis in the presence of sodium dodecyl sulfate, composed of 2.55% polyacrylamide crosslinked with 2.75% methylenebisacrylamide, is described. This gel has a resolving power for very high molecular weight proteins and can be stained with silver. The gel is suitable for fractionation of factor VIII/von Willebrand factor directly from plasma samples. Visualization by silver staining revealed a series of covalently bound multimers with molecular weights of up to 8 X 10(6). The procedure described should be useful also for studies on other very high molecular weight proteins and nucleic acids.     

Modified method of silver staining of proteins in polyacrylamide gels

The very sensitive and reliable silver staining method to visualize proteins in polyacrylamide gels described by Wray et al. (Anal. Biochem. (1981) 118, 197-203) fails when the protein sample contains nucleic acids and/or metals. By washing the polyacrylamide gels in acetic acid and repeatedly in methanol immediately following electrophoresis and then using the procedure of Wray et al., many gels otherwise unstainable may be stained with a high degree of reliability. This method allows visualization of a minute amount of proteins in samples containing high amounts of DNA and metals.