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PubMed1985

Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing.

Polyacrylamide gels were stained with the sialidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid showing the activity of Vibrio cholerae and Clostridium sordellii sialidases in the gels after electrophoresis. With this fluorogenic method minimum sialidase activities of 5 microU could be determined. The sensitivity of this staining is about 10,000-fold higher compared to protein-staining with Coomassie brilliant blue. For the visualization of other proteins than sialidases the specific sialidase staining could be followed by a protein-staining method in the same gel.

Chemical Phenomena↗
PubMed1985

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.

Chemical Phenomena↗
PubMed1985

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.

Carbohydrates↗
PubMed1985

A method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose.

An improved procedure for the electrophoretic transfer of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose is described. The use of more alkaline transfer buffers and the omission of an equilibration step before the transfer allow for the almost complete transfer of strongly basic proteins from gels to nitrocellulose without lowering the transfer efficiency for other proteins.

Buffers↗
PubMed1985

Fluorescent properties of histone-1-anilinonaphthalene 8-sulfonate complexes in the presence of denaturant agents: application to the rapid staining of histones in urea and Triton-urea-polyacrylamide gels.

In the present report it is shown that histone bands in urea-acetic acid or Triton-urea-acetic acid-polyacrylamide gels can be stained with the fluorescent dye 1-anilinonaphthalene 8-sulfonate and visualized by transillumination of the gel with an uv-light source. The 1-anilinonaphthalene 8-sulfonate staining method described here for urea and Triton-urea gels is rapid (it can be completed in 90 min) and allows the detection of less than 1 micrograms of histone per band.

Anilino Naphthalenesulfonates↗
PubMed1985

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.

Acetonitriles↗
PubMed1986

A method for the direct measurement of glycogen synthase activity on gels after polyacrylamide gel electrophoresis.

A method for the detection of glycogen synthase activity after nondenaturing polyacrylamide gel electrophoresis is described. After the electrophoretical run, the gels were incubated in situ with UDP-glucose and glycogen. Labeled or unlabeled UDP-glucose could be used, since similar activity patterns were obtained by autoradiography or iodine staining of the gels. The method here described offers several advantages in terms of speed, sensitivity, and economy when compared with other procedures.

Animals↗
PubMed1986

Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap.

The membrane trap is a new device for the electroelution of all kinds of charged macromolecules from gels. Instead of dialysis membranes, the membrane trap uses a new membrane. Retention of macromolecules in an electric field by dialysis membranes depends on the presence of sodium dodecyl sulfate (SDS) in the buffer. The new membrane retains all charged macromolecules larger than approximately 5000 Da without adsorbing them, independent of the use of SDS. Here we report the electroelution of five different lipophilic membrane proteins (33 to 193 kDa) of Mycoplasma pneumoniae from preparative SDS-polyacrylamide gels into a 300-microliter recovery volume. After an 8-h elution period, recovery ranged from 80 (193 kDa) to 97% (33 kDa). The "losses" were generally due to proteins still remaining in the gel slice. All of the eluted proteins tested in a dot-blot assay proved to be antigenically active. The advantages of the device described here are easy handling (insertion of membranes, open system), quantitative recovery, and high reproducibility of the elution results.

Antigens, Bacterial↗
PubMed1986

Two-dimensional electrophoresis of yeast histones on cellogel strips and polyacrylamide gels.

For a quick analytical identification of histones a two-dimensional electrophoretic system has been developed. First the proteins are separated on cellulose acetate strips in alkaline buffer. Then they are reelectrophoresed in a second dimension on polyacrylamide gels either with sodium dodecyl sulfate or in the presence of urea at pH 2.7. The technique is demonstrated with yeast and calf thymus histones.

Animals↗
PubMed1986

A sensitive staining technique for the detection of phosphohydrolase activities after polyacrylamide gel electrophoresis.

A sensitive colorimetric method for the determination of Pi in the range 0.5-10 nmol has been adapted for detection of several phosphohydrolase activities in polyacrylamide gels. This procedure, which leads to the formation of a malachite green-phosphomolybdate complex, may be used with many commonly studied enzymes, such as acid and alkaline phosphatases, nucleotidases, and ATPases. Since detergents do not interfere with color development, this assay is useful for monitoring the activity of detergent-solubilized membrane enzymes as well as normally soluble enzymes.

Animals↗
PubMed1986

Purification and analysis of mitochondrial membrane proteins on nondenaturing gradient polyacrylamide gels.

A nondenaturing gradient polyacrylamide gel electrophoresis method is described for the resolution of membrane proteins. Bovine heart inner mitochondrial membranes were solubilized in Triton X-100 and individual complexes were identified by staining for activity and protein. Succinate dehydrogenase was isolated by band excision and shown by electrophoresis under denaturing conditions to be highly purified. In addition, the electrophoretic transfer of NADH dehydrogenase to nitrocellulose was demonstrated. The enzyme was identified on the resulting blot by activity staining and the binding of monospecific antibodies.

Animals↗
PubMed1986

A longitudinal slicer for obtaining multiple uniform slices from cylindrical polyacrylamide gels.

Construction of a longitudinal gel slicer for polyacrylamide gels is described. The apparatus produces intact, undamaged, and identical slices with rectangular cross section from gels of different thicknesses and different concentrations of acrylamide. When an extract containing brain proteins was electrophoresed on a cylindrical gel and then sliced and colored by Coomassie blue, an identical band pattern was obtained in all slices, indicating the absence of any gel distortion during cutting.

Animals↗
PubMed1986

An exponential gradient marker for use with minigel polyacrylamide electrophoresis systems.

Minigel electrophoresis has reduced the required sample size and cost of running polyacrylamide gels without loss of resolution. The many problems incurred in the minigel system when pouring multiple gradient gels are eliminated by pouring individual gels with the described economical, exponential gradient marker, which can accurately deal with the small gel volumes. The advantages of exponential gradients are discussed.

Buffers↗
PubMed1986

Electrophoresis of 35S-labeled proteoglycans on polyacrylamide-agarose composite gels and their visualization by fluorography.

We have developed techniques for the electrophoresis of 35S-labeled proteoglycans on polyacrylamide-agarose gel slabs and subsequent fixation, impregnation, and fluorography of such electrophoretograms. The procedure permits the examination of newly synthesized proteoglycan subspecies using a rapid technique, previously unavailable for these labeled molecules.

Adolescent↗
PubMed1986

Requirement for cysteine in the color silver staining of proteins in polyacrylamide gels.

To determine whether cysteine residues have a contribution to the mechanism of color silver staining, we silver stained sodium dodecylsulfate polyacrylamide gel electrophoresis separations of proteins which have few or no cysteines. Proteins without cysteine stained negatively (yellow against a yellow background) with silver. Proteins with one or more cysteines stained orange, red, brown, or green/gray depending on the mole percentage of cysteine and whether they contained covalently attached lipids. The colors could not be correlated with the mole percentages of cysteine of these proteins indicating that some components other than cysteine affect the staining color of cysteine-containing proteins. Silver staining of amino acids, sugars, nucleotide bases, or lipopolysaccharide dot-blotted onto nitrocellulose paper implicated adenine, lipids, the basic amino acids, and glutamine, but not sugars or other amino acids in silver/protein complexes.

Bacterial Outer Membrane Proteins↗
PubMed1986

Visualization of bilin-linked peptides and proteins in polyacrylamide gels.

Biliproteins and bilipeptides subjected to discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of zinc acetate form a complex which fluoresces an orange color when viewed under ultraviolet light. The complex between the bilin chromophore and the zinc ion fluoresces at wavelengths which can be selectively visualized in gels by using a red filter. For the biliproteins phytochrome and C-phycocyanin the minimum detectable quantities are 100 and 50 ng, respectively. This is comparable to the sensitivity of Coomassie blue staining. The technique has been used for selective detection of phytochrome in plant extracts and to distinguish chromophore-bearing peptides from those not containing chromophore in proteolytic digests of phytochrome.

Bile Pigments↗
PubMed1986

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.

Catalase↗