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At least 1,207 records · Page 67Linked to original sources
PubMed1980

Gel gradient electrophoresis, isoelectric focusing and two-dimensional techniques in horizontal, ultrathin polyacrylamide layers.

An ultrathin layer, horizontal polyacrylamide gel system for electrophoresis, isoelectric focusing and two-dimensional techniques is described. Gel slabs 240 micron thin for unidimensional, or 360 micron thin for two-dimensional runs are cast on cellophane foils as support. The sample is loaded in pockets pre-cast in the gel (2--3 microliter size) or in trenches for two-dimensional experiments. The second dimension is routinely performed in concave exponential gel gradients, spanning an acrylamide concentration from 4% to 22.5%. The sensitivity with the common Coomassie Blue stain is very high, well below 0.1 microgram protein/band. Zymogram detections can be developed within a few minutes, thus retaining the band sharpness of the focused zones or of the bands separated in pore gradient electrophoresis. Sample handling, staining and destaining and gel drying and storage are greatly simplified and performed in a fraction of the time needed for conventional, thick gels in the 1-2 mm thickness range.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

The use of prontosil for the visualization of carbonic anhydrase bands in polyacrylamide gels. Application to the carbonic anhydrase isozymes of erythrocytes and white skeletal muscle of the rabbit.

Prontosil, a carbonic anhydrase inhibitor of orange-red colour, is used to visualize carbonic anhydrase bands during isoelectric focusing in polyacrylamide gels. 5-60 ng of the sulfonamide Prontosil are added to the 100-200 microliters samples before application to the gels. Bound Prontosil moves into the gel together with carbonic anhydrase and stains the enzyme bands formed there, while unbound Prontosil remains on top of the gels. The method is specific, no proteins other than carbonic anhydrase were observed to be stained, and it requires no special equipment. It was applied to chloroform/ethanol extracts of erythrolysates and white muscle homogenates from rabbits. Densitometric evaluation of the Prontosil-stained bands obtained with these extracts showed that rabbit red cells contain roughly equal amounts of carbonic anhydrase isoenzymes B and C while in rabbit white skeletal muscle isoenzyme C is predominant and little B enzyme occurs. These results confirm previous findings obtained by affinity chromatography of erythrolysates and muscle homogenates.

Animals↗
PubMed1983

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices.

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.

Acholeplasma laidlawii↗
PubMed1985

Combination of three techniques for the study of complex enzyme mixtures: polyacrylamide gel electrophoresis, immunodiffusion and detection of enzymatic activity.

A technique is presented in which two different separation methods are combined. The first separation is carried out in polyacrylamide gel electrophoresis, which is then followed by immunodiffusion in agarose against appropriate antisera. Soluble and insoluble macromolecular carbohydrates were used as substrates in the detection of the various enzymatic activities. These three methods in combination can provide new information on enzymatically active proteins in mixtures, such as isoenzymes, and multiple forms varying in molecular size.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel. A facilitated method for multi-spot analysis of electrograms.

Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels. Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h. Staining of E. coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well. The absorbance spectra of the complexes in the gel matrix differ significantly from each other. However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Separation of gliadin at pH 3.1 in a polyacrylamide gel suitable for blotting procedures.

A polyacrylamide gel electrophoresis system for the separation of gliadin at acidic pH is described. The gel is cast at neutral pH with polymerization in 20 min. Equilibration of the gel to pH 3.1 takes place during the electrophoresis. The gel is highly uniform with good mechanical properties and therefore suitable for blotting procedures.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

pH, urea and substrate gradients for the optimization of ultrathin polyacrylamide gel zymograms.

The preparation of ultrathin polyacrylamide gels with different kinds of gradients (pH, substrates, inhibitors) is described. By using these gels for contact printing after isoelectric focusing with Ampholines or Immobilines and for diffusion tests, the influence of pH or increasing amounts of substrates or inhibitors on enzyme and isoenzyme activities is studied. These methods are successfully applied for the optimization of zymogram techniques and for the easy characterization of industrial microbial enzyme preparations for technological purposes. With buffer-generated pH gradient gels, the pH optimum of all isoenzyme activities is demonstrated by contact printing; the total amount of isoenzyme activities dependent on pH is determined by a diffusion test. Gels with a linear gradient between 0 and 8 M urea are used for isoelectric focusing, diffusion tests and contact printing in order to differentiate the unfolding and denaturing effects of urea on isoenzymes. Alterations in polygalacturonase isoenzyme patterns dependent on urea concentration are not caused by inhibition or denaturation but by the change of charges. In respect to band sharpness and straightness urea can be added advantageously up to 2 M without changing the isoelectric points or activities of the isoenzymes. For the reproducibility of zymograms it is interesting to see that different substrate concentrations reveal different isoenzyme patterns.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Casting immobilized pH gradients into cylindrical polyacrylamide gels.

A novel method is described for casting immobilized pH gradients in polyacrylamide gel rods of small diameter (2 mm), based on the principle of rotational centrifugation. The tubes are filled vertically with equal volumes of dense and light solution (250 microliter each) titrated to the extremes of the desired pH gradient, and then tilted at 2.5 degrees to the level. After 5 min at rest, to allow for sliding of the two menisci to equilibrium position, the glass tubes are rotated for 3 min at 180 rpm, followed by an additional 3 min at 180 rpm by reversing the sense of rotation. A homogeneous linear gradient is thus produced. The rotating platform is then raised to 90 degrees and the gels allowed to polymerize under standard conditions. Formation of linear and reproducible pH gradients is ensured by using stabilizing density gradients of low viscosity (0-5% glycerol, having a maximal ratio viscosity/density of 1.1).

Acrylic Resins↗
PubMed1988

Enhancement of polyacrylamide gel slice dissolution in hydrogen peroxide by cupric sulfate.

An improved method is described for quantitation of radio-labelled protein by scintillation counting after polyacrylamide gel electrophoresis. The method is based upon copper catalyzed dissolution of gel slices in hydrogen peroxide under ambient conditions. Complete dissolution of gel sections was accomplished by incubation at 25 degrees C in 30% H2O2 that contained 0.9 mM CuSO4. Recovery of tritiated protein was greater than 90% under these conditions while in the absence of CuSO4 recovery was less than 50%.

Acrylic Resins↗
PubMed1991

Transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride membranes.

We have developed a method to transfer proteins from a silver-stained polyacrylamide gel to a polyvinylidene difluoride (Immobilon-P) transfer membrane (Millipore, Bedford, MA). If the silver stained gels are rinsed in 2 x SDS Laemmli sample buffer prior to transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transferred to a single sheet of Immobilon-P without a prior rinse in sample buffer. Most important in the Western blot the antigenicity of the transferred protein is retained in either way. The method described is simple, inexpensive and versatile. A slight modification of the technique permits one to extract minor proteins, or detect their antigenic activities, without contamination of contiguous proteins.

Blotting, Western↗
PubMed1991

Assay for protease inhibitors: qualitative measurement and detection in polyacrylamide gel electrophoretograms.

A simple and fast method for qualitative measurement of protease inhibitors has been developed. The high sensitivity of the assay is based on a casein-precipitating reaction at pH 5.5. Cellulose paper strips, moistened with protease inhibitor-containing solutions, are applied to the surface of an agar-skim milk gel previously coated with a protease solution. After incubation for 2 h at 37 degrees C, inhibitory activity is indicated by a translucent zone (absence of casein degradation) on a white background of precipitated small casein fragments. In addition to being useful for rapid screening of a large number of complex samples, the method allows the localization of protease inhibitor activity in sodium dodecyl sulfate-polyacrylamide gel electrophoretograms.

Agar↗
PubMed1993

Sensitive silver-staining detection of bacterial lipopolysaccharides in polyacrylamide gels.

For sensitive detection of bacterial lipopolysaccharides in the nanogram range, three almost identical silver-staining methods are often used, which are based on ammoniacal silver solutions and an acidic developer. We modified a method used for proteins, based on neutral silver nitrate solution and an alkaline developer, for the visualization of lipopolysaccharides in polyacrylamide gels, which yields better sensitivity and is much less prone to formation of non-specific background staining than the acidic developer-based silver stains.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

The interference of cysteine thiols in the detection of glycated and non-glycated proteins by modified silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

In the previous study a staining intensification of in vitro glycated collagen type I versus a non-glycated one after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under a modified silver staining procedure was observed (Hodny, Z., Struzinsky, R. and Deyl, Z. (1992) J. Chromatogr. 578, 53-62). While investigating the specificity of this stain to glycation product(s) on protein we have observed that a great number of proteins (e.g. bovine serum albumin) was sensitive to this stain even in a non-glycated state. It is proposed from results of the analysis of amino acid composition of these proteins that their better stainability correlates with the amount of cysteine present in the protein. Modification of SH groups by iodoacetamide (or N-ethylmaleimide) had an inhibitory effect on the staining of bovine serum albumin (and some other proteins) in its 'native' state but had no visible inhibitory effect on their staining in the glycated state. However, the positive staining response of a great number of components from cellular lysates even after iodoacetamide treatment indicates the existence of further chemical groups (either of protein or nucleic acid origin) participating in this silver staining method.

Collagen↗
PubMed1995

Fluorescent staining for proteins on polyacrylamide gels with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride).

A simple and sensitive post electrophoresis fluorescent staining technique for proteins on polyacrylamide gels using 5-dimethylamino-1-naphthalene sulfonyl chloride (dansyl chloride) has been developed. Dansyl chloride staining increases the sensitivity, 0.125 micrograms protein per band can be visualised by this technique. The staining method appears to be applicable to all types of proteins including proteoglycans.

Dansyl Compounds↗
PubMed1994

A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels.

A simple and rapid protocol for silver staining of proteins following electrophoresis in polyacrylamide gels (PAGE) is described. We have reduced the number of steps in the procedure of Blum et al. (Electrophoresis (1987) 8, 93-99), and shortened fixation and washing times so that efficient detection of proteins can be achieved within 30 min. In common with more time-consuming silver-staining methods, the present protocol is capable of detecting nanogram quantities of proteins on a colorless background and is suitable for rapid screening of large numbers of samples.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Origin of multiple bands of proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis--intermolecular disulphide cross-linking due to the presence of oxidizing components in the reducing agent.

The presence of oxidized products in the reducing agent used in sodium dodecyl sulphate-polyacrylamide gel electrophoresis is shown to yield multiple bands from otherwise homogeneous RNase A. The role of oxidized products in generating multiple bands is elucidated by using varying proportions of oxidized and reduced glutathione in a mixture as a reducing agent.

Cross-Linking Reagents↗