Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine

A new fluorescent prestaining method for gel‐separated glycoproteins in 1D and 2D SDS‐PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time‐consuming steps needed for poststains. As low as 4–8 ng glycoproteins (transferrin, α1‐acid glycoprotein) could be selectively detected, which is comparable to that of Pro‐Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis was performed to confirm the specificity of the newly developed method.     

Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.     

Method for rapid removal of sodium dodecyl sulfate from polyacrylamide gels

A method is described for the isolation of hepatic microsomes by polyethylene glycol 6000 fractionation of the postmitochondrial fraction of liver homogenate. The procedure is simple and rapid requiring two centrifugation steps at 8000g for 10 min. The preparation has peptide patterns and levels of drug metabolic and other enzymatic activity similar to those of the microsomal fraction isolated by high-speed centrifugation and is referred to as polyethylene glycol 6000 microsomes. It is clarified with detergents and can serve as the starting material for the purification of microsomal proteins.     

Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses, particularirly in the study of the allergenic response to Parietaria judaica pollen in senescence, for Southern-blot hybridizations and in the study of DNA polymorphisms.     

Chromatographic recovery of polypeptides from copper-stained sodium dodecyl sulfate polyacrylamide gels.

A procedure of preparative electrophoresis is described in which proteins separated on sodium dodecyl sulfate gels, stained with copper and eluted by simple diffusion, are highly concentrated on a fluorocarbon packing and freed of small molecular weight substances, including sodium dodecyl sulfate and buffer components and gel-related substances. This method can be used for microscale preparations or it can be scaled up to recover milligram amounts of protein. The purified polypeptides, however denatured, are suitable for amino acid sequencing.     

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels

The use of 3,3′,5,5′-tetramethylbenzidine-H₂O₂ as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.     

Electrophoretic patterns of differently prepared fibrinogen subunits in sodium dodecyl sulfate containing polyacrylamide gels

The electrophoretic behaviour of mercapto, carboxamidomethyl, carboxymethyl and thiosulfonic acid derivatives of rabbit fibrinogen subunits was investigated electrophoretically in sodium dodecyl sulfate containing polyacrylamide gels. Comparing carboxymethyl, carboxamidomethyl and thiosulfonic acid derivatives with the corresponding mercapto subunits divergent electrophoretic patterns were observed. Especially, the position of the Bbeta-chain was strongly dependent on the method of preparation. Similar results were obtained from investigating electrophoretic mobilities of albumin with differently substituted SH-groups after reduction with mercaptoethanol.     

Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins.

We report here a modification to copper and zinc chloride staining methods. The introduction of a preincubation of the gels, prior to metal staining, with 0.2 M imidazole allows the formation of a homogeneous background for the subsequent precipitation of the metal chelate. The reported imidazole-zinc staining takes minutes, resulting in reproducible staining patterns with only slightly lower sensitivity than silver staining. The method allows efficient recovery of proteins from previously stained gels and is compatible with immunoidentification on Western blots and also with amino acid analysis and NH2-terminal sequence analysis of transferred proteins. A mechanism is proposed to explain the observed improvement in reproducibility and sensitivity of imidazole preincubation to zinc staining.     

Localization of cellular antigens in sodium dodecyl sulfate- polyacrylamide gels

A procedure is described for localizing antigen-antibody complexes in sodium dodecyl sulfate (SDS) polyacrylamide gels using 125I-labeled protein A from Staphylococcus aureus. We use the procedure to probe antigenic cross-reactivities between Strongylocentrotus and Chlamydomonas alpha- and beta-tubulins; we also domonstrate how the procedure can detect minor antibody species in an antiserum directed against a cell membrane.     

A method for in-gel fluorescent visualization of proteins after native and sodium dodecyl sulfate polyacrylamide gel electrophoresis

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation–emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.     

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.     

Aberrant migration of lipopolysaccharide in sodium dodecyl sulfate/polyacrylamide gel electrophoresis

Purified lipopolysaccharides of salmonellae strains were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Pre-electrophoresis of polyacrylamide gels had no apparent effect on one-dimensional silver-stained lipopolysaccharide profiles. However, without pre-electrophoresis, two-dimensional and three-dimensional patterns contained numerous bands with varied migration patterns compared to those in the one-dimension gels. The lipopolysaccharide was altered within the polyacrylamide gel during electrophoresis. Pre-electrophoresis of gels eliminated aberrant migration patterns.     

Improving sodium dodecyl sulfate polyacrylamide gel electrophoresis detection of low-abundance protein samples by rapid freeze centrifugation

This work presents a rapid and simple freeze centrifugation method to concentrate dilute protein solutions for detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) Coomassie blue staining. Moreover, a simple way to assemble a cryoconcentration device is presented, and its use is discussed. Commercial purified protein standard and an enzyme with high fructosyltransferase (FTase) activity, coming from target fractions obtained by chromatographic separation, were used as an example. FTase, coming directly from the chromatographic fractions, was difficult to view through SDS–PAGE analysis; however, it was easily visualized, and its activity was enhanced, after the application of the freeze centrifugation protocol presented here.     

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.     

A simple and rapid method for the isolation of peptides from sodium dodecyl sulfate-containing polyacrylamide gels

A rapid and simple method is described for the recovery of peptides from sodium dodecyl sulfate-containing polyacrylamide gels. It involves the electrophoretic concentration of a peptide in the stacking gel followed by elution into glycerol. The method requires no special equipment or chemicals, and the elution can be made using the same electrophoretic systems used in the separation step. The method is more rapid than normal extraction procedures, and simpler than most electrophoretic elution methods described. The method can be used for isolation of microgram as well as milligram quantities of an individual peptide with yields of approximately 100%.     

Resolution of high-molecular-weight components in lipopolysaccharides of Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus strains with sodium dodecyl sulfate polyacrylamide gels

The use of 0.5% sodium dodecyl sulfate in polyacrylamide separation gels allowed the resolution in several bands of high-molecular-mass components in smooth lipopolysaccharide of bacterial outer membrane from Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus. With or without 0.1% SDS, however, such a result was not possible.     

Stability of aprA-subtilisin in sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on the structure and activity of aprA-subtilisin, a secreted bacterial serine protease which is 85% homologous to subtilisin BPN', was examined. The addition of SDS resulted in the slow conversion of the subtilisin from the intact protein to the completely unfolded form of the enzyme. No intermediates between these two populations were detected. This conversion was accompanied by decreased activity, disruption of tertiary structure, a change in the mobility of the protein when subjected to SDS-polyacrylamide gel electrophoresis, and an increase in the apparent Stokes radius of the protein. After 2 h in 1% SDS at 20 degrees C, 25% of the subtilisin was still intact and active. The amount of protein existing in the unfolded form was increased by increasing the length of time in SDS, by increasing the concentration of SDS, and by increasing the temperature of the subtilisin-SDS solution. Analysis of the dependence of the rate of unfolding on SDS concentration indicated that one SDS micelle can destroy two protein molecules. The activation energy for the SDS-induced denaturation of aprA-subtilisin was 20 kcal mol-1, indicating that unfolding of the protein could be the rate-limiting step.