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.     

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).     

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.     

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.     

Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels

A system was developed for the rapid characterization of microbial pectic enzyme complexes and then tested on Erwinia chrysanthemi and Sclerotium rolfsii. Pectic enzymes in minute samples of crude culture filtrates were resolved by ultrathin-layer polyacrylamide gel isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then assayed with an ultrathin pectate-agarose overlay stained with ruthenium red. The simple procedure can be completed within 30 min after isoelectric focusing, can detect extremely low levels of pectate lyase (6.4 x 10 mumol of product per min), and is sufficiently sensitive to determine the pectate lyase isozyme profile of a single bacterial colony with a diameter of 4 mm. Pectate lyases and polygalacturonases can be distinguished by altering buffer conditions in the overlays. The assay system revealed additional isozymes not resolved by classical techniques and generally corroborated the previously published isoelectric points and molecular weights of the pectate lyase isozymes and exo-poly-alpha-d-galacturonosidase produced by E. chrysanthemi and the endopolygalacturonase and exopolygalacturonase produced by S. rolfsii.     

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.     

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.     

Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels

A method for determining which molecules in a complex mixture of proteins can function as bacterial receptors was devised. Salivary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Bacteria that were metabolically labeled with 3H or externally labeled with 125I were incubated on the nitrocellulose replicas. After 18 h at 4 degrees C, the unbound cells were removed by repeated washing of the replicas, and the bands to which the radiolabeled bacteria bound were visualized by autoradiography. By this technique, Fusobacterium nucleatum, which adheres via carbohydrate residues on receptor molecules, and Staphylococcus aureus, which recognizes the peptide portion of fibronectin, were shown to bind specifically to their respective receptors. These results suggest that this method can be useful for profiling bacterial binding to either the carbohydrate or the protein portions of molecules present in complex mixtures, such as those composing biological fluids or tissue substrates. Structural specificities, such as recognition sequences formed by certain oligosaccharides, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N- and O-linked carbohydrate substituents that could serve as bacterial receptors.     

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.     

Sialoglycoproteins with a high amount of O-glycosidically linked carbohydrate moieties stain yellow with silver in sodium dodecyl sulfate-polyacrylamide gels

Several sialoglycoproteins and human salivary proteins were analyzed in sodium dodecyl sulfate-polyacrylamide gels using the silver/Coomassie-staining protocol (J. K. Dzandu, M. E. Deh, D. L. Barratt, and G. E. Wise, 1984, Proc. Natl. Acad. Sci. USA 81, 1733-1737) to determine the extent to which yellow Ag staining originally reported for human red blood cell glycophorins can be applied to other sialoglycoproteins. Results showed that not all sialoglycoproteins elicit a positive yellow color in the silver stain reaction. Some of the sialoglycoproteins stained as brown or negative images in the Ag-staining cycle. Alkaline beta elimination of O-glycosidically linked carbohydrate chains of glycophorin resulted in the loss of yellow color development in the Ag-staining protocol. Analysis of acidic salivary proteins showed several yellow Ag-stained bands at Mr X 10(-3) = 150, 82, 70, 51, 46, and 42. These results suggest that the carbohydrate moieties of glycophorin removable by alkaline beta elimination are responsible for the characteristic yellow color in the Ag stain reaction. In addition, under our staining conditions sialoglycoproteins with a high amount of O-glycosidically linked carbohydrate chains give a characteristic yellow silver stain.     

Ion-enhanced fluorescence staining of sodium dodecyl sulfate-polyacrylamide gels using bis(8-p-toluidino-1-naphthalenesulfonate)

A method for the sensitive fluorescent staining of sodium dodecyl sulfate (SDS) gels that extends the applicability and sensitivity of existing procedures has been developed. SDS-protein complexes are able to bind the noncovalent hydrophobic probe, bis(8-p-toluidino-1-naphthalenesulfonate) (bisANS) with an increase in quantum yield that is considerably larger than that observed with the commonly used monomeric form, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). The quantum yield of bisANS bound to the SDS-protein complex is greatly enhanced by incubation with one of a number of cations including potassium and barium. The use of bisANS with metal ion enhancements provides a method for staining SDS gels that can be more sensitive than commonly used methods based on the binding of Coomassie blue, and provides a simple and rapid method for the detection and quantitation of proteins. The use of metal ion enhancements also greatly increases the sensitivity of staining methods based on the use of 1,8-ANS. The present method is much more sensitive than previous noncovalent, flourescent, postelectrophoresis stains, but it retains their considerable advantages of speed, simplicity, and the ability to perform secondary procedures on the separated materials.     

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].     

Crossed immunoelectrophoresis from sodium dodecyl sulfate-polyacrylamide gels after fixation and staining and without nonionic detergent intermediate layers

A modified method is described for crossed immunoelectrophoresis in which the first-dimension separation has been carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The described method does not require nonionic detergents and is carried out after fixation and staining of the polyacrylamide gel. This permits more precise alignment of immunoprecipitates with polypeptide bands as well as allowing direct testing of an individual polypeptide band for reaction with antibody.     

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.     

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.     

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.     

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.     

SDS聚丙烯酰胺凝胶电泳快速染色新方法的研究

通过几种金融盐溶液对SDS聚丙烯酰胺凝胶电泳染色的实验表明,0.25mol/L的CaCl2和MgCl2溶液能够对蛋白质进行有效的染色,经这2种溶液染色的蛋白质都能够从凝胶中洗脱回收。尤其是CaCl2法灵敏度更高,而且蛋白质条带形成之后也十分稳定,所以在运用制备电泳纯化蛋白质时这种新的染色方法较适用。     

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.