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PubMed2000

Extraction and characterization of adenovirus proteins from sodium dodecylsulfate polyacrylamide gel electrophoresis by matrix-assisted laser desorption/ionization mass spectrometry.

A new methodology for the extraction and characterization of proteins from Coomassie-stained sodium dodecylsulfate polyacrylamide gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been described. The utility of this methodology was demonstrated in the characterization of adenovirus proteins. The key steps in the extraction and destaining process involve washing the excised band with a combination of solvents that include 10% acetic acid, acetonitrile, methanol, and formic acid:water:isopropanol mixture. By using this procedure, we determined adenovirus proteins with molecular weights ranging from 10,000 to 110,000 Da by MALDI-MS, obtaining a detection limit of approximately 6 pmol. Parallel experiments were successfully carried out to analyze adenovirus proteins from Cu-stained gels. It was observed that increase in laser intensity resulted in significant improvements in the quality of MALDI mass spectra for the analysis of inefficiently destained proteins from Cu-stained gels.

Adenoviridae↗
PubMed1999

Electrospray mass spectra from protein electroeluted from sodium dodecylsulfate polyacrylamide gel electrophoresis gels.

Electrospray ionization/tandem mass spectrometry of proteins separated on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels is severely limited by the requirement that the protein be completely separated from the SDS. As shown here, the gaseous noncovalent SDS adducts of protonated proteins thus formed can be dissociated by infrared irradiation. ESI spectra from myoglobin in SDS-containing solutions show molecular ions adducted with up to 15 dodecyl sulfates, but ir irradiation of these ions causes complete dissociation to expel the SDS.

Animals↗
PubMed1999

Observation of gel-induced protein modifications in sodium dodecylsulfate [corrected] polyacrylamide gel electrophoresis and its implications for accurate molecular weight determination of gel-separated proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOFMS) can potentially provide accurate molecular weight information of proteins separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Several issues related to resolution and accuracy of molecular weight measurement are investigated by using a time-lag focusing MALDI-TOF mass spectrometer. The effects of the gel components SDS, glycerol, and tris buffer on the mass spectral signals are studied systematically. Glycerol and tris buffer are shown to have little or no effect on resolution and mass accuracy, whereas SDS degrades sensitivity, resolution, and mass accuracy even at low concentrations. A simple and fast gel extraction technique is presented which is capable of detecting proteins loaded at the low-picomole level on the gel. The sample preparation procedure used in this work appears to remove most of SDS from the gel, thereby reducing the peak broadening effect caused by SDS and resulting in high resolution and accurate measurement of proteins. However, for proteins containing cysteines, the molecular ions are composed of a distribution of acrylamide-protein adducts likely formed by reaction with unpolymerized acrylamide in the gel during the gel separation process. The implications of gel-induced protein modifications on the accurate molecular weight measurement of gel-separated proteins are discussed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

Monomeric inhibitors of influenza neuraminidase enhance the hemagglutination inhibition activities of polyacrylamides presenting multiple C-sialoside groups.

BACKGROUND: Influenza viruses use hemagglutinin (HA) arrays to bind to sialic acid moieties on the surface of cells; crosslinking of erythrocytes by this mechanism leads to hemagglutination. A number of synthetic polymers containing multiple sialic acid (Neu5Ac) groups as side chains are potent inhibitors of this process. Inhibition may be due to two mechanisms: polyvalent binding of the inhibitor's multiple Neu5Ac side chains to multiple HA sites on the viral surface, or steric stabilization of the viral particle by a layer of the adsorbed, water-swollen polymer, which prevents adhesion to the erythrocyte. The balance between these two effects is not yet known. RESULTS: Polyacrylamides with multiple C-sialosides (PA(Neu5Ac)) were 2-20 fold more effective as inhibitors of virally mediated hemagglutination when assayed in the presence of Neu2en-NH2, a potent monomeric inhibitor of influenza neuraminidase (NA). The ability of monomeric inhibitors of NA to enhance the inhibition of hemagglutination in this assay correlated with the affinity of the monomer for NA. CONCLUSIONS: We propose that inhibitors of NA act by competing with the C-sialosides of PA(Neu5Ac) for binding to the active sites of the NA. Competitive displacement of Neu5Ac causes an expansion of the layer of polymeric gel absorbed to the virus, enhancing its inhibitory effect. This study provides an example of synergy between two ligands directed toward the active sites of two different proteins, and reinforces the conclusion that steric stabilization is important for the activity of polyvalent inhibitors.

Acrylic Resins↗
PubMedDate not supplied

Haptoglobin typing of bloodstains using horizontal discontinuous polyacrylamide gel electrophoresis.

A horizontal electrophoresis method using a discontinuous polyacrylamide gel to identify haptoglobin polymorphs is described. Improvements over the traditional vertical gel method include less sample required, easy gel preparation, and shortened run time. Additionally, vertical electrophoresis equipment, which can be technically challenging, is not needed. Caucasian, Black and Hispanic databases, each created from 100 autopsy blood samples from the New York City Medical Examiner's Office, are also presented.

Blood Grouping and Crossmatching↗
PubMed2003

Catalogue of soluble proteins in human vitreous humor by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry including seven angiogenesis-regulating factors.

A catalogue of proteins in the human vitreous humor may contribute to elucidating the pathogenesis of various diseases in ophthalmology. To improve the recovery of proteins in vitreous, we applied one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE). Proteins were extracted from unstained gel strips and digested in gel with trypsin and the peptides were analyzed by capillary-column reversed-phase high-performance liquid chromatography coupled with electrospray ionization-ion trap-mass spectrometry. From a patient with diabetic retinopathy, 84 different proteins were identified. Most of the proteins which we identified in vitreous previously using 2D-PAGE were also identified in the present study. In total, we identified 121 different proteins including five proteins seen at the genomic level only. Four angiogenic factors, insulin-like growth factor, vascular endothelial growth factor, fibroblast growth factor, and placental endothelial cell growth factor, and three anti-angiogenic factors, pigment epithelium-derived factor, endostatin, and thrombospondin, were found, and this may contribute to elucidating the pathological changes in the concentration and the modified structures of these proteins, in diseases of the retina, especially, diabetic retinopathy.

Amino Acid Sequence↗
PubMed1976

The quantitative measurement of whey proteins using polyacrylamide-gel electrophoresis.

A method is described for the quantitative estimation of a mixture of whey proteins by spectrophotometric scanning of the stained protein bands following polyacrylamide-gel electrophoresis. The incorporation of beta-lactoglobulin A as an internal standard improves the accuracy of the technique. The method has been used to measure residual native whey proteins in milk after heating. Results are presented for whey protein denaturation in skim-milk after heat treatment at 130 and 140 degrees C for various periods of time.

Animals↗
PubMed1997

Proteinases secreted by Fasciola hepatica: time course of the inhibitory effect of serum from experimentally infected rabbits demonstrated by gelatin-substrate polyacrylamide gel electrophoresis.

Fasciola hepatica secretes proteolytic enzymes to aid it to penetrate and migrate through the host tissues. Two of these proteinases have been purified and shown to be cathepsin L-like, and are termed, CL1 (27.5 kD) and CL2 (29 kD). The immunogenicity of these proteinases was investigated over the course of an experimental infection and following drug treatment. Four groups of rabbits were studied: group 1: orally infected with 50 metacercariae; group 2: infected and treated 8 weeks after infection; group 3: infected, treated at week 8 and reinfected at week 13 and group 4: non-infected control group. Sera were collected weekly from each group until week 20 postinfection. CL1 and CL2 were incubated with the different sera and then analysed by gelatin substrate polyacrylamide gel electrophoresis (GS-PAGE). Analysis of groups 1, 2 and 3 showed that CL1 and CL2 neutralizing antibodies appear at week 5 post-infection. In group 1, these remained throughout the 20 weeks of infection. In group 2, neutralizing antibodies disappeared at week 13, that is, 5 weeks after anti-Fasciola treatment. In group 3, CL1 and CL2 neutralizing antibodies disappeared at week 13 but reappeared by week 15, that is 2 weeks after reinfection. Pooled sera from group 4, showed no inhibitory capacity. ELISA results using CL1 and CL2 as antigen, correlate very well with the inhibitiory time course observed by GS-PAGE. These results suggest that purified cathepsin Ls are antigenic molecules recognized early in the infective process and capable of inducing a specific humoral response, strong enough to neutralize, at least partially, their enzymatic activity.

Animals↗
PubMed1983

An analysis of the proteinases of Trichomonas vaginalis by polyacrylamide gel electrophoresis.

The proteinases of Trichomonas vaginalis have been analysed by electrophoresis in polyacrylamide gels containing denatured haemoglobin. Seven bands of activity were detected indicating multiple proteinases. All of the enzymes were stimulated by 1 mM dithiothreitol and had inhibitor sensitivities characteristic of cysteine proteinases. The enzymes differed significantly, however, with respect to pH optima and relative sensitivities to inhibitors.

Animals↗
PubMed1989

Characterization of coccidial proteins by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Two dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D SDS-PAGE) has been used to produce 'fingerprint' maps of the proteins from each of the 7 species of Eimeria which infect the chicken. All 7 species could be identified from their array of polypeptides but few differences were detected between strains of the same species. Alterations to the polypeptide array associated with the stage of sporulation of the oocysts were observed. Iodination of sporozoites, 2D SDS-PAGE, autoradiography and immunoblotting techniques were combined to identify polypeptides with a surface moiety and those which were antigenic.

Animals↗
PubMed1991

Protein analysis of Theileria sergenti/buffeli/orientalis piroplasms by two-dimensional polyacrylamide gel electrophoresis.

Proteins of the piroplasms of Theileria sergenti, T. buffeli and T. orientalis were analysed by two-dimensional polyacrylamide gel electrophoresis. Protein spot patterns of T. buffeli and T. orientalis were identical except for a few minor proteins, whereas spot patterns of two T. sergenti stocks were differentiated from those of T. buffeli and T. orientalis by a characteristic set of proteins including a major protein of molecular weight 33-34 kDa. This result indicates that Japanese T. sergenti can be phenotypically distinguishable from European and Australia Theileria species; T. orientalis and T. buffeli.

Animals↗
PubMed1988

Analysis of Opisthorchis viverrini antigens by immunoprecipitation and polyacrylamide gel electrophoresis.

Antigenicity of adult Opisthorchis viverrini, metacercariae and eggs was investigated by radio-immunoprecipitation and polyacrylamide gel electrophoresis using sera from patients with opisthorchiasis and from hamsters experimentally infected with O. viverrini metacercariae. 125I-labelled aqueous somatic extract and in vitro metabolic products of adult worms, and the extracts of metacercariae and eggs were immunoprecipitated, electrophoresed and autoradiographed and major antigens were characterized. Although components with Mr of greater than 116, 89, 78 and 20 kD appeared to be specifically associated with the somatic extract of adult fluke, the 89 kD protein was the most prominent component found in the in vitro culture fluid of adult worms and the metacercarial extract. Hamsters, experimentally infected with O. viverrini, also showed the presence of antibody reactive with the 89 kD component within 15 days of infection. The potential of this antigen in immunodiagnosis is discussed.

Animals↗
PubMed1992

Specific base recognition of oligodeoxynucleotides by capillary affinity gel electrophoresis using polyacrylamide-poly(9-vinyladenine) conjugated gel.

Poly(9-vinyladenine) was synthesized and utilized as an affinity macroligand entrapped within the gel matrix. Base-specific separation of oligodeoxynucleotides was achieved with high resolution and high speed by electrophoresis, using capillaries filled with conjugated polyacrylamide-poly(9-vinyladenine) gel. Oligothymidylic acids were selectively separated from the mixture of oligothymidylic and oligodeoxyadenylic acids by utilizing a specific hydrogen bonding between poly(9-vinyladenine) and oligothymidylic acids. Migration time and resolution of oligodeoxynucleotides were influenced by several parameters, such as the size of poly(9-vinyladenine), capillary temperature, and concentrations of poly(9-vinyladenine) and urea. Some guidelines are presented, based on the theoretical formulation of the effect of these parameters, in order to find optimum electrophoretic conditions. Analytical capillary affinity gel electrophoresis was developed for the selective and sensitive base recognition of oligodeoxynucleotides with efficiencies as high as several 10(6) plates/m by using a urea-gel capillary with poly(9-vinyladenine) and temperature-programming.

Adenine↗
PubMed1992

Separation and identification of DNA-carcinogen adduct conformers by polyacrylamide gel electrophoresis with laser-induced fluorescence detection.

We have developed a separation protocol utilizing high-resolution polyacrylamide gel electrophoresis (PAGE) to isolate stable anti-benzo[a]pyrene diol epoxide adducts of oligodeoxynucleotides. Both enantiomers produced multiple adduct species. The distribution of adduct types could be quantitated by densitometry of autoradiograms or Cerenkov counting of eluted oligomers modified by anti-BPDE isomers. Laser-induced fluorescence (LIF) spectra of eluted adducts at 4.2 K (fluorescence line-narrowing spectroscopy) and 77 K revealed that bands corresponded to pure conformers of pyrene chromophore. Carcinogen-modified oligodeoxynucleotides were single-stranded, but there were often considerable stacking interactions between the pyrenyl residues and the oligonucleotide bases, indicating that electrophoresed oligomers were single-stranded but in a native, versus random coil, conformation. The ability to identify and quantitate adducts by PAGE-LIF, coupled with the high resolution and sensitivity of both techniques, makes PAGE and LIF in tandem a potentially powerful tool in the study of chemical carcinogenesis or other ligand-DNA interactions.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1994

Separation of fragments up to 570 bases in length by use of 6% T non-cross-linked polyacrylamide for DNA sequencing in capillary electrophoresis.

Non-cross-linked polyacrylamide is a very convenient medium for the separation of DNA sequencing fragments in capillary electrophoresis. We demonstrate DNA sequencing with this matrix at an electric field of 200 V/cm and at room temperature. Resolution is observed to decrease exponentially with fragment length. Fragments 570 bases in length generate a resolution of 0.5, which is adequate for sequence identification.

Cross-Linking Reagents↗
PubMed1995

Polyacrylamide-based redox polymer for connecting redox centers of enzymes to electrodes.

Enzyme electrodes based on complexing a water-soluble copolymer of acrylamide and vinylimidazole with [Os(dmebpy)2C1]+/2+ (dmebpy = 4,4'-dimethyl-2,2'-bipyridine) and cross-linking with oxidases by water-soluble cross-linkers are described. The potential of the polyacrylamide-based redox polymer is +55 mV (SCE), a typical electron diffusion coefficient (De) in the redox hydrogel that results from its cross-linking is (1.3 +/- 0.1) x 10(-9) cm2/s. The properties of the enzyme electrodes formed when this redox hydrogel "wired" horseradish peroxidase (HRP), lactate oxidase (LOx) or glucose oxidase (GOx) depended on the thickness of the hydrogel film, the chemistry of their cross-linking, and their enzyme content. At the wired HRP electrodes, H2O2 was electrocatalytically reduced to water at 0.0 V (SCE). Lactate and glucose were electrocatalytically oxidized at 0.16 V (SCE). The GOx electrodes, when made with 140 micrograms/cm2 thick polymer films, were selective for glucose in the presence of physiological concentrations of urate and ascorbate.

Acrylic Resins↗
PubMed1995

Use of non-cross-linked polyacrylamide for four-color DNA sequencing by capillary electrophoresis separation of fragments up to 640 bases in length in two hours.

Four-color DNA cycle sequencing was performed on an M13mp18 template using dye-labeled primers. Sequencing fragments were separated by capillary electrophoresis at 60 degrees C and at an electric field of 150 V/cm. The sieving medium was 5%T, non-cross-linked polyacrylamide in 7 M urea. The use of high temperature for the separation reduces formation of secondary structures in the sequencing fragments, generating a sequence that is free of compressions without the use of strongly denaturing gels. The use of high temperatures also increases the separation rate compared with room-temperature operation. Fragments up to 640 bases are separated in less than 2 h.

Base Sequence↗