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PubMed2005

Application of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides for analysis of hyaluronan and chondroitin sulfate in human and animal tissues and cell cultures.

Hyaluronan (HA) and chondroitin sulfate (CS) are glycosaminoglycans (GAGs) with great importance in biological events, since they participate in and regulate cell adhesion, migration and proliferation. Quantitation and analysis of the fine structure of GAGs are increasingly important for understanding many biological processes, among which are many critical aspects of pathology development and specific phenotype descriptions. Human umbilical cord and human synovial fluid are connective tissues containing high amounts of GAGs and change in the quantity and structure of these macromolecules is described in tissue development and is commonly associated with disease. Moreover, also in Xenopus laevis embryo development and chondrocyte cultures, the GAG content and structure play a critical role. A rapid analysis of hyaluronan and chondroitin sulfate Delta-disaccharides derived from the above human and animal samples, derivatized with 2-aminoacridone and analyzed by polyacrylamide gel electrophoresis, is described in this report. Qualitative and quantitative analysis were performed by comparing their migration and the pixel density with standard Delta-disaccharides, running in the same gel. Since this method allows the analysis of large numbers of samples simultaneously in one gel and has a relatively high sensitivity (less than 25 pmol), it is suggested as a cost-effective and useful tool for the fast screening of small amounts of hyaluronan and chondroitin sulfate disaccharides.

Aminoacridines↗
PubMed2006

Identification of the commonest cystic fibrosis transmembrane regulator gene DeltaF508 mutation: evaluation of PCR--single-strand conformational polymorphism and polyacrylamide gel electrophoresis.

In the present study we investigated whether single-strand conformational polymorphism (SSCP) and polyacrylamide gel electrophoresis (PAGE) could be used for the identification of the CFTR DeltaF508 gene mutation, which is commonest in the Greek population. Using DNA from patients carrying this mutation, the appropriate 98 bp region of the CFTR gene was amplified by PCR and the reaction products were analysed by non-radioactive SSCP-electrophoresis using silver staining for band visualization and non-denaturating PAGE to confirm the results. SSCP electrophoretic analysis has been optimized for several parameters in order to achieve the best resolution. Single-strand DNA fragments gave a reproducible pattern of bands, characteristic for the particular mutation. Comparison of the obtain patterns with control samples allowed the detection of the DeltaF508 mutation in the patients studied by SSCP assay and these results were confirmed by the independent method of PAGE. Although SSCP and PAGE can be used for detection of this mutation, PAGE resulted in more distinct patterns than SSCP. It is, therefore, proposed that PAGE can be reliably used for the detection and identification of such a mutation in patients provided that suitable controls are available. The applicability of PAGE to identification of the mutation in carriers, particularly useful for population screening, is also discussed.

Cystic Fibrosis↗
PubMed1994

Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search.

Mass spectrometric peptide mapping of proteins separated by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis has been investigated. The best results are obtained after blotting of the proteins onto polyvinylidene difluoride membranes followed by enzymatic digestion of the protein on the membrane. The peptide maps were investigated in terms of completeness and applicability for protein identification using a previously developed database search program as well as for the possibility for full characterization of covalent modifications in the proteins. The most complete peptide maps were obtained when the proteins were reduced and alkylated on the membrane prior to enzymatic digestion followed by separation of the resulting mixture by high performance liquid chromatography prior to mass spectrometric analysis. Such peptide maps cover up to 98% of the sequence and consequently may allow complete characterization of post-translational modifications in proteins for which the amino acid sequence is known. The fastest and most sensitive procedure to obtain peptide maps sufficient for protein identification was direct analysis of the extracted peptide mixture by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The use of external and internal calibration of MALDI spectra for database searches is evaluated as well as the possibility of including a post-calibration routine within the search program.

Alkylation↗
PubMed1984

Helper T cell activation for the human B cell response to trinitrophenylated polyacrylamide beads: involvement of the T4 antigen.

The monoclonal antibody (mAb) OKT4A (but not OKT4) inhibits the in vitro antibody response of human peripheral blood mononuclear cells. The target of OKT4A mAb is the helper T cell, as the helper cells for the antibody response to trinitrophenylated polyacrylamide beads (TNP-PAA) are exclusively in the T4 subset. The OKT4A mAb is still suppressive when the anti-TNP response of cultures of monocyte-depleted cells is supported by purified interleukin 1. Both the OKT4A mAb and anti-DR mAb suppress the non-specific T cell proliferation in the cultures leading to the in vitro mAb response. A parallel inhibition is observed for the autologous mixed lymphocyte reaction, the nonspecific B cell response, but the T cell response to mitogens is not affected. These results suggest that the recognition of self major histocompatibility complex class II determinants by the T4 molecule plays a major role in the activation of T helper cells for antibody production to this particulate antigen.

Acrylic Resins↗
PubMed1988

Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem Development Unit.

Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate-polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

A continuous acetic acid system for polyacrylamide gel electrophoresis of gliadins and other prolamines.

A polyacrylamide gel electrophoresis system buffered by acetic acid alone was developed for electrophoresis of prolamines. When applied to gliadin electrophoresis, the acetic acid system produces more bands than does a conventional aluminum lactate-lactic acid system (using 12% acrylamide gels). The acetic acid system is relatively simple, requiring a single buffer component that is universally available in high purity.

Acetates↗
PubMed1988

High resolution two-dimensional electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cheese proteins after rapid solubilisation.

The proteins of cheese are rapidly solubilised by heating to 95 degrees C in buffered 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol. Electrophoretic analysis of the solubilised proteins by either one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis or high resolution two-dimensional electrophoresis yields reproducible patterns characteristic of an individual cheese and its extent of ripening. The patterns reveal (i) the residual amounts of milk casein and whey proteins, and (ii) the appearance of casein degradation products, including pink-violet components as detected by Coomassie Blue staining.

Cheese↗
PubMed1988

Improved transfer of high molecular weight proteins using fabric-reinforced soft polyacrylamide gels.

Rapid transfer of electrophoretically separated, high molecular weight proteins from fabric-reinforced, soft, low-concentration polyacrylamide gels (3.5%T, 2.6%C) is described. Polyester fabrics proved useful for gel reinforcement during vertical slab gel electrophoresis. The fabric-reinforced gel retains its dimension during transfer, equilibration and drying, facilitating comparison of protein bands in the separation gel and transfer matrix.

Collodion↗
PubMed1988

Ferguson plots based on absolute mobilities in polyacrylamide gel electrophoresis: dependence of linearity of polymerization conditions and application to the determination of free mobility.

In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacrylamide gel electrophoresis based on their absolute mobilities were found to be linear under unusual polymerization conditions which yield relatively wide gel fibers and a low total fiber length per unit weight, but not under previously and commonly used conditions. These linear Ferguson plots in gels of 1, 3 and 5% crosslinking intersect at a single gel concentration between 1 and 2% T (M-point). It is postulated that the measure of free mobility of the proteins is the M-point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously. This postulate abolishes the well-known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo) in terms of free mobility. The postulate is also compatible with the interpretation of the points of intersection of the Ferguson plots of oligomeric series of proteins at finite gel concentrations (designated mu-points) as their common free mobilities.

Buffers↗
PubMed1988

Rapid imaging, using a cooled charge-coupled-device, of fluorescent two-dimensional polyacrylamide gels produced by labelling proteins in the first-dimensional isoelectric focusing gel with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone.

A new method for visualising proteins in two-dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) while present in the first-dimensional gel after isoelectric focusing and subsequently electrophoresed into the second-dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge-coupled-device was used to view the two-dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two-dimensional gels at the end of electrophoresis with no further processing.

Bacterial Proteins↗
PubMed1988

Blotting from PhastGel media after horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

An improved procedure, "thermoblotting", is described for transferring proteins by diffusion from PhastGel Gradient media to an immobilizing matrix after horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis the gels were left on the separation bed of PhastSystem, the blotting matrix was applied and a transfer temperature was selected between 5-70 degrees C. An experimental series at fixed diffusion times showed that the transfer yield was significantly increased with temperature. The evaluation was done visually after staining of the blots with colloidal gold. An evaluation study comparing nitrocellulose, nylon, and polyvinylidenedifluoride of different pore sizes is also reported. Finally, the transfer efficiencies for 125I-labelled bovine serum albumin and soybean trypsin inhibitor were estimated using four different blotting procedures: two diffusion blotting techniques and two electrophoretic blotting techniques (tank vs. semi-dry).

Collodion↗
PubMed1988

The use of ultrathin-layer polyacrylamide gel isoelectric focusing in two-dimensional analysis of plant and fungal proteins.

Although use of ultrathin polyacrylamide gel isoelectric focusing in the first dimension of two-dimensional analysis bestows a number of advantages, it has been little used by the plant science community. Nonstandardization along with problems unique to the format have probably delayed wider adoption. Relevant parameters were therefore tested in order to optimize resolution, reproducibility, economy and ease of use. Ultrathin-layer gels (200 microns in this study) used in the first dimension require a semirigid backing for support. Widely available matte-finished thin polyester film without chemical pretreatment was found to bind the gel adequately. The gel adheres to the film through all processing steps, yet, if desired, can be easily transferred to Whatman 3MM paper for special applications such as Western blotting. The ultrathin first-dimensional gels can be quickly dried on the polyester backing for convenient handling and long term storage. Strips cut from the dried gel for use in the second dimension are more easily manipulated than their tube format counterparts. The difficulty of disrupting and recovering microsamples of labeled leaf and root tissue prompted the invention of an efficient and simple communition device. An economical and efficient silver stain process is also described. This analytical technique was applied in an attempt to detect resistance gene products in different genetic backgrounds of maize. Although the ultrathin flatbed format provides as good as or better resolution than the tube gel system, the level of sensitivity was still inadequate to reveal the apparently rare resistance gene product.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Polypeptide patterns obtained by polyacrylamide gel electrophoresis of thylakoid membranes isolated from light and dark grown strains of Chlorogloea fritschii.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis has been used to determine the difference between thylakoid polypeptide patterns of light and dark grown strains of the cyanobacterium Chlorogloea fritschii. There were only 2 prominent bands present in the dark grown strains, polypeptide Mr50,000 and polypeptide Mr90,000, also five fainter bands in the Mr range 45,000-66,200 corresponding to photosystem one, compared with the 32 bands present in the light grown strains. There was no obvious indication of the Mr 33,000 3-(3,4-chlorophenyl)-1,1-dimethyl-urea binding protein. In addition the progressive daily development of the various photosystem components in the light and their relationship in photosynthesis was determined. It was observed that the increase of the relative concentrations of the photosystem two and phycocyanin components indicated their developments are mutually synchronized. The effect of light to dark and dark to light transfer on established strains was investigated. Appreciable loss of photosystem two components and the presence of an additional band Mr22,500 of unknown function in the light to dark transfer, and little reactivation of the photosynthetic capabilities in the dark to light transfer was observed.

Cell Membrane↗
PubMed1989

Effect of electric field switching on the electrophoretic mobility of single-stranded DNA molecules in polyacrylamide gels.

We have examined the effects of pulsed electric fields on the separation of single-stranded DNA molecules in polyacrylamide sequencing gels. Using different electric field pulsing regimens, the mobilities of single-stranded DNA molecules can be retarded or increased as compared to conventional electrophoresis. These results indicated that pulsed field techniques can be applied to gel electrophoresis of small single-stranded DNA molecules.

DNA, Single-Stranded↗
PubMed1989

Use of a polynomial exponential function to describe migration of proteins on sodium dodecyl sulfate-polyacrylamide gels.

Standard mixtures of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polynomial regression analysis was used to fit curves to the data points obtained by plotting log10 of protein molecular weight versus electrophoretic mobility. Polynomials with orders ranging from 1 to 4 were generated. The coefficients of each equation were analyzed for statistical significance. It was found that a third order polynomial was the highest-order equation in which all coefficients contributed significantly to the prediction of molecular weights. Using this equation, it was possible to estimate the molecular weights of known proteins in the range from 97,400 to 14,400 with a maximum error of 1%, compared with a maximum error of 17% when a first-order equation was used to describe the migration of the standards.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

The application of sodium dodecyl sulphate-polyacrylamide gel electrophoresis to the taxonomic identification of the total body protein band profiles of Diplostomum spp. metacercariae (Digenea), parasites of fish eyes.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis has been used to determine the metacercariae (Mc) total body protein band profiles of different Diplostomum spp. Four species of fish were investigated, roach (Rutilus rutilus) infected with D. spathaceum Mc, gywniad (Coregonus laveratus) infected with D. coregonus Mc, ruff (Gymnocephalus cernua) infected with a Diplostomum species related to D. gasterostei Mc, and perch (Perca fluviatilis) infected with D. gasterostei Mc. The four species of Diplostomum Mc were distinguished by three different bands of molecular weight, Mr 55,500, 53,500 and 52,000. A homology of polypeptide component distribution was evident for D. gasterostei Mc, from P. fluviatilis, and D. coregonus Mc, from C. laveratus, the latter showing reduced protein concentrations between bands Mr 32,000-40,000. Critical analysis of Mc polypeptide patterns showed no evidence of contamination with lens, retina or vitreous humour host eye protein. Gross morphological data for the parasites was also considered in relation to different band profiles obtained for the Diplostomum spp. Mc. Band profile analysis in conjunction with other taxonomic tests proved to be a useful tool in the identification of unknown species.

Animals↗