Identification and subgrouping of Frankia strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Polypeptide patterns of soluble proteins from 35 Frankia strains from different plants of various geographical origins, belonging to Alnus and Elaeagnus host-specificity groups were determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide pattern was qualitatively the same for each strain whatever the number of subcultures or the age. Two gel electrophoresis groups A and E were observed which matched with the Alnus and Elaeagnus host-specificity groups, but with some exceptions. The polypeptide patterns of the 35 Frankia strains tested were separated into 13 gel electrophoresis subgroups. Five Frankia strains were inoculated separately or in 3 mixed combinations of 2 strains on Alnus glutinosa (L.) Gaertn. plants. The polypeptide patterns of the re-isolates obtained from 5-month-old nodules were identical to the corresponding strains used initially in the inoculum. Dual infection was observed on single plantlets.     

One-step casting of Laemmli discontinued sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel

A modified Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol is described. The new method saves 30 min for gel casting without loss of the resolution power of Laemmli gel. In this method, both the upper and lower gels can be cast at the same time because the lower gel contains 10% glycerol, which generates higher density in the lower gel than in the upper gel.     

A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described. The slab get consists of two porous layers of acrylamide of the following composition: 4% acrylamide, 0.04% bisacrylamide for the stacking gel, and 10% acrylamide, 0.1% bisacrylamide for the separating gel, both layers having different buffers. The separating gel mixture (final pH 9.0) and the buffers of the electrode chamber (pH 8.45) consist of Tris and glycine in such a ratio that no acid or base is necessary to adjust the pH. The resulting gel system has the following advantages: (a) it is able to resolve the components from large-volume samples (up to 200 microliter) after an overnight electrophoresis run while still maintaining the capacity to produce very sharp bands; (b) it has a high and broad resolution, allowing the separation on the same gel of proteins with apparent molecular masses between 10,000 and 450,000 Da; (c) it is very easy to prepare and shows excellent reproducibility in the electrophoretic patterns; (d) when used as a second dimension in tandem with isoelectric focusing, it improves the resolving power of two-dimensional gel electrophoresis; and finally, (e) its low crosslinker-to-acrylamide ratio allows the effective and rapid transfer of proteins to nitrocellulose membrane, thus improving the usefulness of protein blotting. In all cases, adrenal medullary chromaffin cell proteins were used as test samples.     

Mobility of acetylated histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We describe an altered mobility for acetylated histone isoforms in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoforms of histones H3 and H4 with a higher acetylation degree have a slightly faster electrophoretic mobility. Since acetylation neutralizes the positive charge of the epsilon-amino group of lysine, without significantly changing the molecular mass of the protein, the acetylation-dependent mobility shift could be explained by the increase of the net negative charge of the SDS-histone complexes. A possible consequence of this differential mobility for the acetylation site determination by protein microsequencing from SDS gels is discussed.     

Modification of a discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide gel system for minigel electrophoresis

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system was recently described by J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271). The system was developed to separate with high and broad resolution the components from large volume samples after an overnight electrophoresis. This system was found to have many advantages. However, when used directly as a minigel system, this method cannot sustain the high voltage inherent to minigel electrophoresis and produces artefacts, namely a double front and a loss of resolution in the low molecular weight range. These problems were eliminated using the buffer system of M. A. Porzio and A.M. Pearson [1977) Biochem. Biophys. Acta 490, 27-34) in the separating gel and in the electrode chambers. The resulting modified discontinuous minigel system has the same advantages as the technique described for large slab gel electrophoresis, including the effective and rapid transfer of high molecular weight proteins to nitrocellulose membranes, as well as the advantages of the minigel format.     

Quantification and partial characterization of the hamster sperm proacrosin-acrosin system

The proacrosin-acrosin proteinase system was measured and partially characterized in unpurified extracts of washed hamster epididymal sperm. Autoactivation experiments demonstrated that proacrosin accounted for greater than 98% of the acrosin activity in the sperm extracts from individual animals. Several bands of proteinase activity were observed on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic (gelatin-SDS-PAGE) zymography. The major proteinase activities in the nonactivated extracts corresponded to relative molecular masses (Mr) of 51,000 to 56,000, while less distinct digestion occurred with relative molecular masses of 37,000 to 49,000. It was demonstrated that after a serial dilution of the sperm extract, the proteinase activity in as few as 6,000 sperm could readily be detected by the gelatin-SDS-PAGE methods. Time-course activation studies showed that the zymogen was completely converted to active proteinase in 45-60 min at pH 8.0 and 25 degrees C. This autoconversion process was markedly inhibited by calcium, sodium, and heparin. However, each of these compounds stimulated the proteolytic activity of acrosin. These studies demonstrate that the proacrosin-acrosin system can be investigated in extracts of nonpurified hamster epididymal sperm.     

Quantitative determination of overlapped proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A method for resolving an overlapped polypeptide pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis was described. The procedure was essentially a Gaussian fitting using the least squares method, and could resolve more than 20 overlapped components simultaneously. The applicability to overlapped and shouldered patterns was evaluated using practical electrophoretic data with varying amounts of mitochondrial samples. The relative contents of respective polypeptide components gave a good agreement regardless of the loaded amounts.     

Lysozyme activity in animal extracts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

1. Lysozyme activity was detected after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels containing 0.2% (W/V) autoclaved Micrococcus lysodeikticus cells as substrate. 2. Lysozyme activity appeared as clear lysis zones after incubation of opaque gels at 37 degrees C in buffered Triton X-100. 3. As low as 0.1 pg of purified hen egg white lysozyme could be detected after 16 hr incubation at pH 6.5. 4. Bands with lytic activity from kidney and pancreas acetone powders, bird's egg whites and vitelline membranes, animal sera and human saliva corresponded to c-type (Mr 14,500), g-type (Mr 20,500) or both lysozymes as far as molecular weight is concerned. 5. Some extracts, like porcine kidney, exhibited more than two bands. 6. Bands with lytic activity migrating at the level of g-type lysozymes were detected in some kidney and pancreas extracts.     

Peptide mapping by CNBr fragmentation using a sodium dodecyl sulfate-polyacrylamide minigel system

The method of peptide mapping by sodium dodecyl sulfate-gel electrophoresis following partial protein fragmentation with cyanogen bromide was adapted for a polyacrylamide minigel system. The combined use of the discontinuous gel electrophoresis system of J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271) and a vertical polyacrylamide minigel system produced the following advantages over other procedures: (a) the ability to resolve cyanogen bromide cleavage fragments over a broad molecular mass range while yielding very sharp protein staining bands; (b) well-defined peptide maps are produced with as little as 2 micrograms of protein; (c) less time is required to perform fragmentation with cyanogen bromide, to equilibrate the gel slices in sodium dodecyl sulfate buffer, as well as to perform the electrophoresis; and (d) the cyanogen bromide fragmentation patterns are highly reproducible.     

Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol

Mercaptoethanol, when present in the sample buffer during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is responsible for the appearance of two nonprotein bands (electrophoretic mobilities corresponding to 68 and 54 kdalton) stainable with silver and with Coomassie blue. After iodination in vitro of DNA preparations isolated by alkaline phenol extraction using chloramine-T procedure, part of the radioactive label is found in these bands, provided the reaction is terminated by mercaptoethanol, whereas only a diffuse background is present in this area if the reaction is stopped by sodium metabisulfite. Similar results are obtained with highly purified total cytoplasmic RNA. The results indicate that the appearance of the 68- and 54-kdalton bands is in artifact. The relevance of these results to the proteins tightly bound to DNA is discussed.     

An evaluation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the identification of microsporidia

Microsporidian spore polypeptides separated with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) can be used to identify isolates of microsporidia. The spore polypeptides separated with SDS/PAGE provided unique, reproducible electrophoretic profiles which were not influenced by host species or the temperature at which the host larvae were maintained for development. Furthermore, host proteins were not detected in electrophoretic profiles of the spore polypeptides. Spore mixtures of two microsporidian species can be detected when the spore polypeptides of either or both species have been previously separated with SDS/PAGE.     

Renaturation of Leishmania donovani 3'-nucleotidase following sodium dodecyl sulfate-polyacrylamide gel electrophoresis

1. Renaturation of a 3'-nucleotidase from the surface membrane of Leishmania donovani promastigotes was achieved following polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). 2. Enzyme activity was detected in situ in gels, following SDS removal, by incubating the gels in reaction mixtures containing 3'-AMP or 3'-UMP as substrate followed by staining for the inorganic phosphate (Pi) reaction product with malachite green-molybic acid solution. 3. Conditions for the removal of SDS by diffusion and for the renaturation of enzyme activity are described including evidence for the detergent requirement, which is best satisfied by 3[(3-cholamidopropyl)-dimethylammonio]2-hydroxy-1-propane sulfonate (CHAPSO). 4. Results indicate that the 3'-nucleotidase migrates under these conditions as a polypeptide with an Mr of 43,000.     

Simple, time-saving dye staining of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue

A fixation-free and fast protein-staining method for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research.     

Electrophoretic mobility of Alzheimer's amyloid-beta peptides in urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The 43-amino acid Alzheimer's amyloid-beta peptide (Abeta peptide) retains a predominantly alpha-helix and beta-strand structure in sodium dodecyl sulfate (SDS) solution. This conformer has a high tendency to aggregate during conventional SDS-polyacrylamide gel electrophoresis (PAGE). Both the secondary structure and the proclivity for aggregation are obviated by the use of urea-SDS-PAGE: In 8M urea-with or without SDS-the Abeta peptide becomes 100% random coil and remains monomeric. However, during electrophoresis in this medium, the peptide and its truncated variants do not obey the law of mass/mobility relationship that most proteins-including Abeta peptides-follow in conventional SDS-PAGE. Rather, the smaller carboxy-terminally truncated peptides migrate slower than the larger full-length peptide, while the amino terminally truncated peptide does migrate faster than the full-length Abeta peptide. Thus, despite their small size (2-4kDa) and minor differences between their lengths, the Abeta peptides display a wide separation in this low-porosity (12% acrylamide) gel. We found that this unusual electrophoretic mobility in 8M urea is due to the fact that the quantity of [35S]SDS bound to the Abeta peptides, instead of being proportional to the total number of amino acids, is rather proportional to the sum of the hydrophobicity consensus indices of the constituent amino acids. It is then their hydrophobicity and, hence, the net negative charges contributed by the peptide-bound SDS that plays a major role in determining the mobility of Abeta peptides in 8M urea-SDS-PAGE. The high selectivity of the 8M urea-SDS-PAGE method allowed us to detect the presence of hitherto unknown Abeta peptide variants that were secreted in the conditioned medium by cultured HeLa cells.     

Sequence homology analysis of a heterogenous protein population by chemical and enzymic digestion using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel system

A procedure for examining possible sequence homology between two or more proteins in a heterogenous protein mixture using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis system is described. Three different chemical reagents (cyanogen bromide, hydroxylamine, and acetic acid) and three enzymes (α-chymotrypsin, trypsin, and Staphylococcus aureus protease) have been used as the cleavage reagents for the peptide mapping studies. Potential application of this technique in conjunction with radioactive labeling and immunological studies was also demonstrated.