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PubMed1989

Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera.

Viral polypeptides were prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by copper staining and electroelution from gel slices. Poliovirus capsid polypeptide VP 1 isolated by this procedure induced monospecific antibodies in rabbits, i.e., antisera reacting only with the homologous polypeptide. Our results demonstrate the applicability of the described copper staining method as a rapid visualization step for preparing viral proteins after SDS-PAGE.

Animals↗
PubMedDate not supplied

The migration anomaly of DNA fragments in polyacrylamide gels allows the detection of small sequence-specific DNA structure variations.

Curved DNA fragments have a reduced electrophoretic mobility in polyacrylamide gels. The retardation in gels is extremely sensitive to small structural variations which influence the DNA helix axis. This gel assay can also be used to detect very small structural variations in DNA sequences which are not curved: The noncurved sequences of interest can be combined with curved stretches in phase with the helix turn. Using such sequence constructions, even subtle influences on the DNA helix axis can be detected. Experiments of this kind allow the determination of a relative order of sequence-specific DNA twist and wedge angles.

Base Composition↗
PubMed1989

Ultrasensitive polychromatic silver staining of sodium dodecyl sulfate-polyacrylamide gels with the Gelcode system using the PhastSystem Development Unit.

The Gelcode color-based silver staining system, an improved formulation based on the original publication by Sammons et al. (Electrophoresis 1981, 2, 141-147) has been adapted to automated rapid staining in the PhastSystem Development Unit. The use of elevated temperatures in the fixation, washing, staining, and stabilization steps of the protocol reduces the total time of the process from 18 h to 1 h. The limit of detection, which is at least tenfold more sensitive than the silver staining protocol recommended for the PhastSystem, corresponds to 0.05-0.1 ng of protein per band. The method is applicable to both one- and two-dimensional polyacrylamide gels.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

A thin-layer multistrip polyacrylamide gel electrophoresis apparatus for Ferguson plot analysis at the submicrogram load level.

A procedure was developed for casting thin-layer multistrip polyacrylamide gels and using them for the simultaneous gel electrophoresis at several gel (Ferguson plot analysis) at the sub-microgram load level, using silver staining, autoradiography and, potentially, blotting for detection. The lower viscosity of polymerization mixtures, compared to agarose gelation mixtures, required the redesign of the multistrip cassette with separation of channels by rubber gaskets and the application of a cassette press. The lowered viscosity also required addition of 35% sucrose and an increased rate of polymerization in application to multistrip gels formed on a common NetFix backing. The present design allows one to obtain Ferguson plots exemplified by those of 32P-labeled DNA followed by autoradiographic detection.

DNA↗
PubMed1990

Reassessment of commercially available molecular weight standards for peptide sodium dodecyl sulfate-polyacrylamide gel electrophoresis using electroblotting and microsequencing.

Myoglobin CNBr peptides, constituting the commercially available molecular weight calibration kits for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were analyzed by microsequencing after electroblotting on polyvinylidene difluoride (Immobilon) membranes. An obvious disagreement was found between peptide identification and the data provided by the manufacturers. We observed 6 peptides from Mr 2500 to 17,000 corresponding, in increasing size order, to the 3 peptides resulting from the total CNBr digestion, to 2 incompletely cleaved peptides and to the intact myoglobin. Using a corrected calibration curve, a linear relationship was established from Mr 6000 to 43,000 and a second one for shorter peptides. This method of electrophoresis and electroblotting, easily adapted for peptides, is a powerful tool for peptide identification correlated with size determination. It is especially useful for CNBr-cleaved peptides.

Amino Acid Sequence↗
PubMed1990

Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.

Ammonia↗
PubMed1990

Two-dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis-related proteins using PhastSystem.

Acidic and basic pathogenesis-related proteins (PR-Ps) were extracted from the intercellular fluid (IF) of soybean leaves, locally infected with tobacco necrosis virus and showing necrotic local lesions. Proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using PhastSystem and precast commercially available gels. Extracts from healthy leaves were run as controls. PR-Ps were first run under native PAGE conditions or isoelectric focusing (IEF), the gels stained with Coomassie Blue, then run under sodium dodecyl sulfate (SDS)-denaturing conditions and finally stained with silver. Ten major acidic PR-Ps were separated; their Mr's were close to those found by conventional PAGE. Their isoelectric points ranged from 3.5 to 5.0. Ten basic PR-Ps were separated and their Mr's estimated. None of these acidic or basic soybean PR-Ps was a glycoprotein. PAGE with PhastSystem and precast gels gives reliable results, comparable with those from conventional 2D-PAGE, with simpler experimental procedures. By electrophoresing Coomassie-stained gels with SDS in the second dimension, we were able to control the first-dimensional separation and to avoid laborious protocols generally adopted with unstained gels.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1990

A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis.

A reddish-brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11-12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2-2 h with 7% acetic acid at 60 degrees C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R-250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity.

Caseins↗
PubMed1990

The application of schlieren optics for detection of protein bands and other phenomena in polyacrylamide gel electrophoresis.

A modernized schlieren optics system was applied to follow protein bands visually during polyacrylamide gel electrophoresis. This approach is suitable for quantitative measurement of electrophoresis as well as for detection of protein bands because of the absence of distortion of the electrophoretic pattern during the staining process. The optics are also suitable for the detection of various background phenomena which usually escape observation.

Buffers↗
PubMed1990

Numerical analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoretic protein patterns for the classification, identification and typing of medically important bacteria.

A numerical analysis of one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoretic protein patterns of three separate bacterial groups is described. Similarity between patterns was estimated using a correlation coefficient and is represented diagramatically by the unweighted pair-group with arithmetic averages method of clustering. Protein patterns were scanned using a laser densitometer interfaced directly to a microcomputer, where calculations were performed semi-automatically with specially written software. A method for the normalization of patterns from different gels is utilized. Whole cell protein patterns clearly distinguished strains representing various groups of Achromobacter. Similarly the two biovars of the Group EF-4 bacteria could be separated after analysis of partial "background patterns" as could the different species of Providencia. In addition, whole patterns could be used to type the various strains of P. rettgeri. These examples serve to illustrate the power, versatility and potential of the technique when applied to problems of classification, identification and typing of bacteria of medical interest. Its advantages and disadvantages, when compared to other available electrophoretic methodologies, are discussed, as is the future application of such technology to the hospital laboratory.

Bacteria↗
PubMed1990

Interlaboratory comparative study of the numerical analysis of one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoretic protein patterns of Campylobacter strains.

Twenty-nine bacterial strains of the genus Campylobacter were examined independently at two collaborating institutes, the National Collection of Type Cultures, London and the Laboratorium voor Microbiologie, Rijksuniversiteit Gent. The one-dimensional polyacrylamide gel electrophoretic protein patterns of the strains were analysed using computerised numerical methods which employed a correlation coefficient and a clustering algorithm. The electrophoretic methods used at the two institutes included both major differences such as gel composition and running conditions and minor differences in buffer composition. Although the algorithm on which similarity and clustering were computed were the same, the detailed treatment of scan patterns differed. The resulting protein patterns in the gels differed markedly in appearance but after numerical analysis the two systems were equally effective in their ability to speciate the strains. There were, however, differences in the relationships between the species defined at the two institutes and these were at least partly due to the different background subtraction methods employed. In conclusion, the portability and reproducibility of the two systems for identification was demonstrated but for definitive classification further standardization may be required.

Bacterial Proteins↗
PubMed1990

Polyacrylamide gel electrophoretic studies on the self-association of crotamine: characterization and molecular dimension of n-mer species.

Polyacrylamide gel electrophoresis (PAGE) at pH 4.4 was used to study the concentration dependence of absolute mobility of crotamine. Within amounts ranging from 5-65 micrograms the toxin appeared in at least three n-mer species which were characterized by their geometrical mean radius R and molecular weight Mr estimation. The R and Mr values of crotamine bands were obtained from equations described in the literature and by using standard polypeptides and proteins submitted to the same experimental conditions. When amounts of up to 20 micrograms were assayed by PAGE the bands had a monomer molecular weight value of 4650 and R was 1.08 nm. From 20-35 micrograms the toxin migrated as dimer (Mr 10,000) with an R value of 1.42 nm. However, amounts higher than 35 micrograms crotamine were mostly resolved in a "two-band" pattern with R and Mr values corresponding to higher associated species.

Calibration↗
PubMed1990

Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein.

Semidry electroblotting is convenient and allows a rapid and efficient protein transfer from one- or two-dimensional polyacrylamide gels onto sequencer stable supports for protein microsequence analysis in a gas-phase sequencer. Using this technique, we determined the amino acid sequences of the basic 7S globulin (Bg), an insoluble protein present in soybean seeds. Based on sequence determination, the cDNA-encoding Bg could be easily cloned and characterized.

Amino Acid Sequence↗
PubMed1990

Calibration of polyacrylamide gel columns for the separation of oligonucleotides by capillary electrophoresis.

Polyacrylamide-filled gel columns are used to separate oligonucleotide samples. For homopolymeric standard samples, plots of migration time versus molecular size are presented over a range of 30-160 bases. With 2.5-4% T and 3.3% C gels, good resolution over the examined mass range, with peak width at half height of 3 to 6 s, is obtained by applying electrical fields of 200-400 V/cm. The separation of heteropolymeric nucleotides by slab gel electrophoresis under routine conditions was compared with capillary gel electrophoresis. Using the same column and the same separation conditions, the plot of migration time versus base number is linear with an identical slope for three oligonucleotide samples which were examined, allowing a calibration of a gel-filled capillary for molecular mass determination.

Base Sequence↗
PubMed1990

Recovery of the contact site A glycoprotein of Dictyostelium discoideum from sodium dodecyl sulfate-polyacrylamide gel and characteristics of monoclonal antibodies against the recovered protein.

Monoclonal antibodies were produced against a cell-cell adhesion (contact site A) glycoprotein of Dictyostelium discoideum, isolated by preparative gel electrophoresis. The glycoprotein was recovered by electroelution from a polyacrylamide gel strip and used for the production of monoclonal antibodies. Four of the five antibodies obtained bound specifically to the protein moiety of the contact site A glycoprotein. The specificities of the antibodies were in striking contrast to those of antibodies raised against the contact site A glycoprotein purified by Triton X-114 phase separation and DEAE chromatography. The majority of the latter antibodies recognized the carbohydrate moiety of the contact site A glycoprotein and cross-reacted heavily with other membrane glycoproteins.

Antibodies, Monoclonal↗
PubMed1991

Structural analysis of recombinant proteins prepared by semi-dry electroblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Proteins that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were electroblotted onto polyvinylidene difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N-terminal sequence analysis. A semi-dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N-Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N-terminal sequence analysis of the purified electroblotted samples. Consequently, time-consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.

Amino Acid Sequence↗
PubMed1991

Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid: application to the enzymological structural analysis of oligosaccharides.

Mono- and oligosaccharides, each containing a reducing end group, were labeled with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid and the resulting derivatives were separated with high resolution by polyacrylamide gel electrophoresis using a method developed recently (P. Jackson, Biochem. J. 1990, 270, 705-713) but with an alternative electrophoretic buffer system. The fluorescent derivatives of glucose and all its straight chain, alpha 1-4 linked, oligomers from maltose to maltoheptaose were well resolved. Various isomers such as maltose and lactose could be separated, as were maltose and cellobiose and some epimers, for instance glucose and galactose. The method was applied to the analysis of the partial sequential degradation of a complex oligosaccharide with neuraminidase and beta-galactosidase. Gels showing fluorescent saccharide band patterns were recorded in picomolar quantities either photographically or using an imaging system based on a cooled charge-coupled device.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Influence of carbohydrate moieties of human serum transferrin on the determination of its molecular mass by polyacrylamide gradient gel electrophoresis and staining with periodic acid-Schiff reagent.

The influence of carbohydrate moieties of transferrin (Tf) on the determination of its molecular mass (MM) by polyacrylamide gradient gel electrophoresis (PAGGE) was investigated. Iron-free native human serum transferrin (Tf) of 99% purity and partly or completely carbohydrate- and N-acetylneuraminic acid (NANA)-free molecule forms were analyzed. The MM differences before and after enzymatic cleavage were found not to agree with the theoretical difference. From amino acid and carbohydrate analysis the MM of Tf was determined to be 79,570 Da whereas by denaturing and nondenaturing PAGGE MM of 77,000 Da +/- 1000 Da were found. After enzymatic cleavage of the two carbohydrate chains of Tf the difference between the calculated MM and the value reported in literature increased to 7000 Da (nondenaturing PAGGE) and 9200 Da (denaturing PAGGE). Following enzymatic cleavage of the 4 NANA molecules (MM 1237 Da) we obtained the relatively largest difference between the value given in the literature and that determined by PAGGE, namely MM 3300 Da on nondenaturing and 4000 Da on denaturing PAGGE. The differences due to the removal of the other carbohydrates were negligible. In addition we tested the periodic acid-Schiff reagent to stain iron-free Tf, containing different carbohydrate residues. The shortest carbohydrate moiety necessary for Tf staining corresponds to two identical carbohydrate chains of the structure (Asn)-GlcNAc-GlcNAc-beta-Man-(alpha-Man-)-alpha-Man.

Amino Acids↗