Rapid fluorescent staining of histones in sodium dodecyl sulfate-polyacrylamide gels

The increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) produced by core histones is higher than that produced by very lysine-rich histones (H1 and H5). In the presence of the anionic detergent sodium dodecyl sulfate (SDS) the enhancement of ANS fluorescence caused by these two groups of histones is roughly the same, but much lower than that observed for core histones in the absence of this detergent. However, the increase of ANS fluorescence produced by histone-SDS complexes is high enough to use it for the staining of these proteins separated in SDS-polyacrylamide gels. Histone bands are stained with ANS after electrophoresis and visualized by transillumination of the gel with a uv light source. The method described in this work allows the rapid detection of less than 0.5 microgram of histone per band.     

Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels

The sensitivity with which RNase and DNase activity can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) varies widely, depending upon the particular SDS preparation used for electrophoresis. (See also [10.], Anal. Biochem. 100, 357–363.) Sensitivity of detection is greatly increased by using buffered 25% isopropanol, rather than buffer alone, to wash detergent from gels after electrophoresis. Thus it is routinely possible to detect bovine pancreatic RNase A at the picogram level. Use of isopropanol improved activity staining of RNases with each of the 10 SDS preparations examined, including one containing 32% tetradecyl sulfate and 4% hexadecyl sulfate, and reduced the variability from preparation to preparation observed when buffer alone was used to remove SDS. Other water-organic cosolvent binary mixtures can be used but none shows advantages over aqueous isopropanol when sensitivity of detection as well as availability and cost of organic solvent are considered.     

Recovery of protein from sodium dodecyl sulfate-polyacrylamide gels

An apparatus for extracting small quantities of protein from sodium dodecyl sulfate-polyacrylamide gels is described. It enables protein contained in a slice of polyacrylamide gel to be transferred electrophoretically, into a small volume of buffer solution. The technique is rapid (within 2 h), reproducible, and efficient (up to 90% recoveries).     

Renaturation of phosphorylase kinase activity from sodium dodecyl sulfate-polyacrylamide gels

Phosphorylase kinase activity is renatured and detected in situ following electrophoresis of the denatured holoenzyme in a sodium dodecyl sulfate-polyacrylamide gel containing phosphorylase b that has been included in the gel polymerization according to the method of R. L. Geahlen et al. [(1986) Anal. Biochem. 153, 151-158]. Among the enzyme's four subunits, only gamma is catalytically active. When extract of rabbit muscle is electrophoresed and renatured in a similar manner, the phosphorylase-conversion activity is also associated only with a protein band that comigrates with the gamma subunit of phosphorylase kinase. This suggests that the gamma subunit of phosphorylase kinase may be the sole activity in rabbit muscle responsible for the phosphorylation of phosphorylase b. In an alternative method for the renaturation of activity from conventional sodium dodecyl sulfate-polyacrylamide gels, the subunits of the enzyme are visualized using 2.5 M KCl, excised from the gel, and eluted by diffusion into buffer containing sodium dodecyl sulfate, which is subsequently removed by acetone precipitation of the eluted subunits. Catalytic activity is recovered when the acetone precipitate of the extracted gamma subunit is dissolved in 6 M guanidine hydrochloride and diluted 50-fold into an activity assay. Inclusion of eluted alpha and beta subunits in the assay inhibits the activity of the gamma subunit, which supports our previous finding that the alpha and/or beta subunits suppress the activity of the catalytic gamma subunit [H. K. Paudel and G. M. Carlson (1987) J. Biol. Chem. 262, 11912-11915].     

Western blots from sodium dodecyl sulfate-polyacrylamide gels stained by metal salts

Proteins separated by sodium dodecyl sulfate-gel electrophoresis can be stained in 5 min with zinc or copper chloride. We here report that these stained but unfixed gels can be electrophoretically transferred to nitrocellulose filters and probed immunologically with the same efficiency and sensitivity as unstained gels. In this way, an immunologically defined polypeptide can be identified with a specific stained protein band on a single gel.     

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.     

Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels

A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.     

Isolation and elution of proteins from sodium dodecyl sulfate-polyacrylamide gels with an intermediate agarose-containing layer in the acrylamide gel

An efficient method for the isolation of a few milligrams of a protein from a protein mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. The method is based on the insertion of an intermediate agarose-containing layer in the polyacrylamide gel. The protein mixture labeled with fluorescamine and the unlabeled one were run simultaneously in separate slots. During electrophoresis the fluorescent-conjugated protein bands were followed by uv illumination. The electrophoresis was stopped when the fluorescent band corresponding to the protein to be isolated was in the agarose layer. The protein is extracted quantitatively from the agarose in less than 1 h by ultracentrifugation. The pure protein recovered in the supernatant was used directly, in the Tris-sodium dodecyl sulfate buffer, to prepare rabbit antiserum.     

Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes.

Two methods are described for detecting less than 1 microgram of highly glycosylated proteins, such as mucins, on sodium dodecyl sulfate-polyacrylamide gels. They combine commonly employed periodic acid-Schiff (PAS) and Alcian blue dyes with silver stain. Carbohydrate prestaining renders mucins more cationic and favors greater complexation with ionic silver. Comparisons of different mucin samples stained either with PAS-silver or alcian blue-silver indicate differential staining between the two techniques. Such differences may, in part, be due to an affinity of Alcian blue for sulfated glycoproteins. These two staining protocols when used in conjunction with silver staining alone are particularly valuable for assessing sample purity and for detecting contaminating proteins during mucin purification protocols.     

In situ detection of beta-lactamase activity in sodium dodecyl sulfate-polyacrylamide gels

After beta-lactamase had been denatured by boiling in the presence of sodium dodecyl sulfate (SDS) and then electrophoresed in SDS-polyacrylamide gels, activity could be restored and could be detected in situ as specific molecular species. Renaturation was simple and facilitated by the presence of a carrier protein. The assay was sensitive, detecting 0.8 ng beta-lactamase activity in the gel.     

Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A rapid and easy method for staining lipopolysaccharides with ethidium bromide is described. Lipopolysaccharides could be visualized by ethidium bromide with almost the same sensitivity as found with the silver-staining method in less than 30 min. The ethidium bromide-staining method was particularly suitable for staining lipopolysaccharides possessing acidic O-specific polysaccharides, which were poorly visualized by silver staining.     

Crossed immunoelectrophoresis from sodium dodecyl sulfate-polyacrylamide gels into antibody-containing agarose: an improved method for evaluation of the number of immunochemical determinants in polypeptides, with reference to spectrin.

A simple method is described for performing crossed immunoelectrophoresis into antibody-containing agarose when the first-dimension gel contains peptides separated by electrophoresis in sodium dodecyl sulfate. Artifacts produced by sodium dodecyl sulfate are avoided by incorporation of Triton X-100 in the agarose layer. Peptides are located by prestaining (before SDS-acrylamide electrophoresis) with the cycloheptylamylose complex of fluorescamine. Injection of ink into prestained peptide bands produces a line extending from the peptide band location to its precipitin arc, thereby allowing unambiguous assignment of arcs to peptides in situations where peptide bands are not widely separated. The utility of this procedure is illustrated for the erythrocyte membrane protein spectrin.     

Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels

We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.     

Affinity purification of polyclonal antibodies from antigen immobilized in situ in sodium dodecyl sulfate-polyacrylamide gels

A procedure for the preparation of affinity-purified antibody is described. Protein mixtures are separated by electrophoresis in sodium dodecyl sulfate (SDS)--polyacrylamide gels. Individual bands of protein are cut from the gel and fixed in situ with glutaraldehyde. The gel pieces are then homogenized and washed extensively with buffered solutions and chaotropic agents. The washed gels can then be used as immunoadsorbents to purify antibodies from crude antisera. This method should be especially useful for the preparation of small amounts of antibody to proteins that are difficult to purify by conventional means, that are available only in limited quantity, or that cannot be blotted to immunoadsorbents such as nitrocellulose or diazotized paper.     

A new method for partial peptide mapping using N-chlorosuccinimide/urea and peptide silver staining in sodium dodecyl sulfate-polyacrylamide gels

A simple, rapid procedure for obtaining partial peptide maps from nanogram quantities of protein in gel slices using the selective tryptophanyl peptide bond cleavage reagent N-chlorosuccinimide/urea is presented. The generated peptide fragments can be visualized by autoradiography or by a sensitive protein silver-staining technique.     

A mass spectrometry compatible silver staining method for protein incorporating a new silver sensitizer in sodium dodecyl sulfate-polyacrylamide electrophoresis gels

A quick, sensitive, and MALDI-TOF MS compatible silver staining method, namely Eriochrome black T (EBT)-silver method, is described. The method can detect 0.05-0.2 ng protein within 60 min in SDS-PAGE gels. EBT dye was used as a silver ion sensitizer having reducing power for silver ions.     

Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels.

In a previous work (J.-R. Daban, M. Samsó, and S. Bartolomé, Anal. Biochem. 199, 162-168, 1991) we observed that, in the presence of the detergent sodium dodecyl sulfate (SDS), diverse types of proteins produced a high increase in the fluorescence intensity of the hydrophobic probe 9-diethylamino-5H-benzo[alpha]-phenoxazine-5-one (Nile red). This enhancement of Nile red fluorescence was observed at SDS concentrations lower than the critical micelle concentration (CMC) of this detergent in the buffer (0.025 M Tris and 0.192 M glycine, pH 8.3) currently used in SDS-polyacrylamide gel electrophoresis. This observation led us to introduce a modification in the typical (U. K. Laemmli, Nature 227, 680-685, 1970) SDS-polyacrylamide gels, in which the SDS concentration in the gel after electrophoresis is lower than the CMC of this detergent but high enough to maintain the stability of the protein-SDS complexes in the bands. The staining of these modified gels with Nile red produces very high fluorescence in the protein-SDS bands and low background fluorescence. The Nile red staining method described in this paper is very rapid (i.e., the bands can be visualized and photographed within 6 min after the electrophoretic separation) and has a high sensitivity, similar to that obtained with the covalent fluorophores rhodamine B isothiocyanate and carboxytetramethyl-rhodamine succinimidyl ester also investigated in this work. Furthermore, our quantitative estimates indicate that most of the protein bands stained with Nile red show similar values of the fluorescence intensity per unit mass.     

Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

This article describes a general method for detecting pegylated proteins directly after SDS-PAGE. The proteins to which polyethylene glycol (PEG) molecules are attached are stained with a barium iodide solution. The staining is based on the formation of a barium iodide complex with PEG. The described method combines a specific staining of PEG molecules with the high resolution of the SDS-PAGE method. It is shown that pegylated protein is detectable on SDS-PAGE as well as on IEF at concentrations that are not detectable by Coomassie protein staining. This paper also describes the determination of the molecular weight of pegylated hirudin by calibrating SDS-PAGE with polyethylene glycol of different molecular weight. Under the conditions used, PEG showed linear mobility during electrophoresis. However, the use of nonpegylated proteins as standards resulted in incorrect molecular weight values due to the lower mobility of the pegylated protein during electrophoresis. The method described might reflect a general method for determining molecular weight of pegylated proteins.     

Fractionation of yeast invertase isozymes and determination of enzymatic activity in sodium dodecyl sulfate-polyacrylamide gels

Three invertase isozymes extracted from derepressed yeast cell membranes with 1% deoxycholate (0.5 mg/mg protein) were separated on sodium dodecyl sulfate (0.25%)-polyacrylamide gels (Babczinski, P., and Tanner, W., 1978, Biochim. Biophys. Acta538, 426–434; Babczinski, P., 1980, Biochim. Biophys. Acta, in press). A dye-producing method for the location of isozymes after gel electrophoresis has been developed by using a coupled enzymatic reaction including the formazan reaction. By the incorporation of β-d-glucose dehydrogenase as the d-glucose-utilizing auxiliary enzyme into the test system the problem of contaminating sucrase activities in commercial glucose oxidase preparations has been overcome (the latter enzyme had often been used earlier in activity-staining procedures of glycosidases). Production of formazan dye is dependent on the presence of sucrose in the test system. By quantitative densitometry proportionality of dye intensity with incubation time, amount of deoxycholate extract, and amount of protein applied onto the gel were determined.