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.     

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.     

A simple and sensitive staining method for the detection of cellulase isozymes in polyacrylamide gels.

A simple and rapid staining procedure is described for qualitative and quantitative determination of the activity of plant (Citrus sinensis (L.) Osbeck cv. Shamouti) and fungal (Trichodermata viride) cellulases in polyacrylamide gels. The method is based on the incorporation of carboxymethyl cellulose, a cellulase substrate, into the gels. After electrophoresis of crude extracts the gels are incubated in sodium-potassium phosphate buffer for the cellulase reaction which is stopped at the desired time by acidification of the gels in 60% sulfuric acid. The gels are then exposed to 2.0% KI + 0.2% I₂. No color develops in areas containing cellulase activity. The experimental procedure is described, and its different aspects are discussed.     

Aspartate aminotransferase isozymes from rabbit liver. Purification and properties

Cytosolic and mitochondrial isozymes of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase [EC 2.6.1.1] ) were purified to homogeneity from rabbit liver. The rabbit liver isozymes were closely similar to the corresponding isozymes from other sources, including human heart, pig heart, chicken heart, and rat liver, in their molecular weights, absorption spectra, amino acid compositions, isoelectric points, and Michaelis constants for the substrates. The NH2-terminal amino acid sequences of rabbit liver isozymes were identified up to 30 residues, and showed some differences from those of the corresponding isozymes obtained from other animals so far studied.     

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 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.     

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.     

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.     

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.     

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.     

Silver staining for selective detection of histones in polyacrylamide gels

A simple silver-staining technique was developed for selective visualization of histones in polyacrylamide gels. The specificity of the stain was confirmed using a variety of protein mixtures and isolated histones. The staining procedure requires a relatively short time to perform (2.5-3 h), and the sensitivity to lysine-rich histones is comparable to that of the conventional Coomassie blue stain (about 0.1 microgram per band). A possible mechanism for the selective staining was deduced from a comparison with the widely used ultrasensitive silver staining.     

Charge-dependent staining of proteins after electrophoretic separation in polyacrylamide gels

A staining procedure is described which allows for the identification of basic and acidic proteins after gel electrophoresis. This includes electrophoresis of sodium dodecylsulfate (SDS) membrane proteins not accessible to isoelectric focusing as a means of charge-dependent separation. Nonfixative charge-dependent staining can be used for detection of proteins after separation in gels, when further investigation of the intact protein is desired.     

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.     

DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.     

A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels

Mycobacteria produce two classes of catalase, designated T and M. Only the T-catalase also has a peroxidase-like function. When a 3,3'-diaminobenzidine (DAB) peroxidase stain was applied to polyacrylamide gel electrophoresis gels, followed by a ferricyanide negative stain for catalase, isoenzymes of T-catalase appeared as dark bands within a zone of clearing in the green background; the M-catalase appeared only as a clear zone. Heated and unheated preparations could be used to demonstrate the presence of comigrating bands of M and T. The application of the ferricyanide stain after the DAB stain of T-catalase resulted in marked intensification of the positive bands of T-catalase due to nonenzymatic, peroxide-independent reduction of the ferricyanide by the DAB product.     

The basis of silver staining of bacterial lipopolysaccharides in polyacrylamide gels

When cast in a polyacrylamide gel, whole lipopolysaccharide (LPS) and the lipid A fraction of LPS fromSalmonella typhimurium andEscherichia coli O111B4 reacted with the silver stain described by Tsai and Frasch [11]. However, the polysaccharide fractions released from the LPS by acid hydrolysis were not stained. This is inconsistent with the previously believed notion that the polysaccharide component is that which reacts with the silver stain.     

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.