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At least 865 records · Page 48Linked to original sources
PubMed1993

Electrospray ionization mass spectrometry on hydrophobic peptides electroeluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis application to the topology of the sarcoplasmic reticulum Ca2+ ATPase.

We describe a method to prepare proteins and peptides in a state suitable for exact determination of molecular mass by electrospray ionization mass spectrometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution. The utility of the procedure, in conjunction with N-terminal sequencing, in defining the C-terminal end of the peptide fragments produced by proteolysis of sarcoplasmic reticulum Ca2+ ATPase with V8 is demonstrated. The application of mass spectrometry aids significantly the use of proteolytic enzymes for topological studies of membrane proteins, and SDS-PAGE is preferable to reverse-phase HPLC for separation of membraneous, hydrophobic peptides and proteins.

Animals↗
PubMed1993

Analysis of receptor-stimulated and basal guanine nucleotide binding to membrane G proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A method to study [alpha-32P]GTP binding to the alpha subunit of GTP-binding proteins in rat brain membranes is described. This method measures receptor-stimulated GTP binding to individual alpha subunits. GTP binding is associated with two protein bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The bands, 40- and 45-kDa in size, comigrate with the alpha subunits of Gi/Go and Gs, respectively. Binding of [alpha-32P]GTP is saturable and Mg(2+)-dependent. Nucleotides compete with [alpha-32P]GTP binding in the following order: GTP > GDP > Gpp(NH)p > App(NH)p. Dopamine stimulates [alpha-32P]GTP labeling of the 40- and 45-kDa bands. A binding increase of 300-400% is observed at 10 microM dopamine. Isoproterenol (10 microM) stimulates [alpha-32P]GTP binding only to the 45-kDa protein band. The effects of dopamine and isoproterenol are blocked by their respective receptor antagonists, fluphenazine and propranolol. The individual G proteins activated by dopamine are resolved by immunoprecipitation of stimulated [alpha-32P]GTP binding to G alpha s, G alpha i, and G alpha o with specific anti-G alpha antisera. Dopamine stimulates [alpha-32P]GTP binding to G alpha s and G alpha i while the labeling of G alpha o was not significantly changed. Pertussis toxin-mediated ADP ribosylation prevents the activation of G alpha i which is mediated by dopamine receptor stimulation. The methods described are useful in defining the coupling of specific neurotransmitter receptors to specific G proteins in native membranes. These procedures also allow measurements of receptor stimulation of individual G proteins in intact biological membranes.

Adenylyl Imidodiphosphate↗
PubMed1993

Sedimentation equilibrium-quantitative polyacrylamide gel electrophoresis (SE-QPAGE): a new technique for the detection of associations in multicomponent solutions.

A new technique for the characterization of self- and hetero-associations in multicomponent solutions without chemical modification of the solutes is described. Solutions containing between one and four distinct protein components were first centrifuged to sedimentation equilibrium in swinging bucket rotors. Following centrifugation, the contents of each centrifuge tube were fractionated into 3-microliter aliquots deriving from sequential laminae of solution, and each aliquot was diluted into sodium dodecyl sulfate-containing buffer. Following incubation, 20 microliters of each aliquot was applied to individual lanes of a polyacrylamide gel. Following electrophoresis, proteins on the gel were stained with Coomassie blue R-250. The pattern of stained bands on the gel was digitized and the resulting image file analyzed to yield relative intensities of individual bands. A quantitative relationship between band intensity and amount of protein in a band was established by means of calibration gels. This relationship was found to be essentially constant for a variety of different proteins except for a protein-dependent scaling factor. Analysis of the dependence of band intensity of a single protein component upon the radial position of the original lamina from which the fraction was derived yielded reliable estimates of the molecular weight of that component in the original solution and in some cases showed that two components were cosedimenting as a complex.

Animals↗
PubMed1994

The analysis of fluorophore-labeled glycans by high-resolution polyacrylamide gel electrophoresis.

Many important biological processes are mediated by the carbohydrate moieties of glycoconjugates. Their structural determination presents considerable difficulties because of both their complex structures and the small quantities which are available from biological sources. In order to address some of the problems in this field a new method of glycan analysis has been developed in which saccharides having a reducing end group are labeled covalently with a fluorophore and the derivatives separated with high resolution by polyacrylamide gel electrophoresis. Fluorophores have been used that enable the electrophoretic separation of positional isomers, anomers, and epimers of a variety of saccharides. Mixtures of complex glycans obtained by enzymatic cleavage from glycoproteins yield electrofluorograms showing high-resolution oligosaccharide profiles. Techniques for obtaining saccharide sequence information from isolated oligosaccharides have been developed. Subpicomolar quantities of individual saccharides can be detected using an imaging system based on a cooled charge-coupled device. The method enables rapid, simultaneous, parallel analyses of multiple samples. The analytical and clinical applications and potential of the system are discussed.

Carbohydrate Sequence↗
PubMed1994

Detection of alginate lyase by activity staining after sodium dodecil sulfate-polyacrylamide gel electrophoresis and subsequent renaturation.

A slab gel electrophoretic method for the study of bacterial alginate lyase has been developed. By incorporating alginate in acrylamide gels, the method is based on renaturation of the enzyme after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent staining with cetylpyridinium chloride, and quantification of the spot by densitometric scanning. The molecular mass of alginate lyase can be determined from its position in the gel.

Azotobacter↗
PubMed1994

N,N'-diethyldithiocarbamate as a stain for copper-zinc superoxide dismutase in polyacrylamide gels of red cell extracts.

N,N'-Diethyldithiocarbamate reacts with copper in the copper-zinc superoxide dismutase (EC 1.15.1.1) in polyacrylamide gels to form stable yellow-brown bands that are quantifiable at 448 nm. This method of examining superoxide dismutase has been applied to crude extracts of the enzyme obtained from red cell lysates from which hemoglobin has been removed by chloroform-ethanol precipitation. This treatment did not affect the activity and heterogeneity of purified dismutase added to lysates and recovered by the same method. The bands that develop in the dithiocarbamate-stained gels of the extracts correspond exactly to the bands of dismutase activity obtained with a positive activity stain using dianisidine, indicating that the dismutase is the only copper protein that gives rise to these bands. The amount of superoxide dismutase in the bands, determined by comparing the areas under unknown peaks to areas obtained with a standard dismutase sample, agrees with the amount predicted from indirect superoxide dismutase activity measurements. Any color in gels due to trace hemoglobin or hemoglobin degradation products is bleached overnight during staining with the dithiocarbamate.

Acrylic Resins↗
PubMed1994

A method for activity staining of peroxidase and beta-1,3-glucanase isozymes in polyacrylamide electrophoresis gels.

A procedure was developed for activity staining of peroxidase and beta-1,3-glucanase isozymes (a double staining procedure) in the same polyacrylamide gels using 3-amino-9-ethylcarbazol for peroxidase staining and laminarine and 2,3,5-triphenyltetrazolium chloride for glucanase staining. The procedure was used to detect peroxidase and beta-1,3-glucanase in different corn lines using plant leaf extract.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Acrylamide in polyacrylamide gels can modify proteins during electrophoresis.

Many notable discoveries have resulted from the characterization or purification of compounds by electrophoresis. The results reported here show that when polyacrylamide gels are used as the support matrix for electrophoresis, proteins can be modified by reaction with unpolymerized monomers of acrylamide, forming covalent acrylamide adduction products. This was demonstrated by electrophoresis of human and rat hemoglobins and subsequent analysis by electrospray ionization mass spectrometry. The degree of covalent acrylamide binding can be appreciable. The degree of adduction is dependent upon the condition of the gel matrix, the amount of material applied, and the reactivity of the material under investigation.

Acrylamide↗
PubMed1994

Electrophoresis for genotyping: microtiter array diagonal gel electrophoresis on horizontal polyacrylamide gels, hydrolink, or agarose.

Electrophoresis of DNA has been performed traditionally in either an agarose or acrylamide gel matrix. Considerable effort has been directed to improved quality agaroses capable of high resolution, but for small fragments, such as those from polymerase chain reaction (PCR) and post-PCR digests, acrylamide still offers the highest resolution. Although agarose gels can easily be prepared in an open-faced format to gain the conveniences of horizontal electrophoresis, acrylamide does not polymerize in the presence of air and the usual configurations for gel preparation lead to electrophoresis in the vertical dimension. We describe here a very simple device and method to prepare and manipulate horizontal polyacrylamide gels (H-PAGE). In addition, the open-faced horizontal arrangement enables loading of arrays of wells. Since many procedures are undertaken in standard 96-well microtiter plates, we have also designed a device which preserves the exact configuration of the 8 x 12 array and enables electrophoresis in tracks following a 71.6 degrees diagonal between wells (MADGE, microtiter array diagonal gel electrophoresis), using either acrylamide or agarose. This eliminates almost all of the staff time taken in setup, loading, and recordkeeping and offers high resolution for genotyping pattern recognition. The nature and size of the gels allow direct stacking of gels in one tank, so that a tank used typically to analyze 30-60 samples can readily be used to analyze 1000-2000 samples. The gels would also enable robotic loading. Electrophoresis allows analysis of size and charge, parameters inaccessible to liquid-phase methods: thus, genotyping size patterns, variable length repeats, and haplotypes is possible, as well as adaptability to typing of point variations using protocols which create a difference detectable by electrophoresis.

DNA↗
PubMed1994

Molecular sieving of polystyrene carboxylate of a diameter up to 10 microns in solutions of uncrosslinked polyacrylamide of M(r) 5 x 10(6) using capillary zone electrophoresis.

Capillary zone electrophoresis of polystyrene carboxylate (PSC) up to 10 microns in diameter in 0.1 to 0.9% solutions of uncrosslinked polyacrylamide (PA) of M(r) 5 x 10(6) demonstrates effective molecular sieving in that medium. The reduced mobility, however, is not constant in Tris-borate-EDTA (1x TBE) buffer containing the polymer but increases in proportion to the load of PSC and inversely to the concentration of PA unless the TBE concentration is increased 10-fold or 50 mM 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid is added to the solution or in the absence of PA. Both the Ferguson plots [log(mobility) vs PA concentration] of PSC obtained at various PSC loads and the effects of the dissociating conditions, high ionic strength and detergent, signify that the variable mobilities are those of different PSC aggregation states. Thus, only the fully associated or dissociated states of PSC provide mobilities that are constant independently of PSC load. The study shows that the information inherent in Ferguson plots regarding particle and fiber properties can be exploited for particles up to 10 microns in diameter by capillary zone electrophoresis in polymer solutions even if these particles form various aggregation states. Thus, such an analysis appears feasible in application to biologically relevant and functional complexes such as subcellular-sized particles or DNA-protein complexes, providing that these particles maintain their integrity and functionality under the selected electrophoretic conditions.

Acrylic Resins↗
PubMed1994

Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.

Electrophoresis of proteins and protein-protein complexes in polyacrylamide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detection of both negatively and positively charged proteins as well as protein-protein complexes in the same gel. During electrophoresis, a continuous gel sandwiched between two glass plates is placed horizontally on the platform and submerged in a reservoir buffer. The sample wells are made along the center of the gel, allowing positively and negatively charged proteins to migrate toward the cathode and anode, respectively. Several proteins with varying molecular weights and isoelectric point (pI) values and pairs of proteins capable of forming protein-protein complexes were chosen as model systems to illustrate the methodology. The effects of several parameters on the performance of the gel system including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, both negatively and positively charged proteins as well as protein-protein complexes can be transferred by electroblotting onto polyvinylidene difluoride membranes for further analyses.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

A specific colorimetric staining method for gamma-carboxyglutamic acid-containing proteins in polyacrylamide gels.

In this paper we describe a specific staining method for gamma-carboxyglutamic acid (Gla)-containing proteins in polyacrylamide gels. The procedure is based on the colorimetric detection of Gla using 4-diazobenzenesulfonic acid and has the advantage of being simple and fast (1 h). The detection limit is 9.4 pmol for prothrombin and 150 pmol for osteocalcin. It is demonstrated that Gla-proteins can be visualized independent of the presence of an excess of contaminating non-Gla-proteins. The technique may be used for screening of large number of fractions during the purification of Gla-proteins from complex protein mixtures.

1-Carboxyglutamic Acid↗
PubMed1995

Modulation of the behavior of a protein in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate by varying the cations.

The denaturation of Aspergillus oryzae alpha-amylase by dodecyl sulfates can be modulated by the change of cations among sodium, lithium, and several alkanolammonium ions. The denaturation can be detected by polyacrylamide gel electrophoresis in the presence of alkanolammonium dodecyl sulfates, particularly around 0 degree C where the denaturation reaction can be virtually frozen. The moderate stability of the amylase against denaturation by dodecyl sulfates helped to demonstrate the difference among the cations. We previously reported a significant difference among the dodecyl sulfates in their ability to dissociate chlorophyll-protein assemblies. The present results indicated that there is also a difference in the ability of the dodecyl sulfates to denature the amylase, a monomeric water-soluble protein. Two species, one retaining intact disulfide bonds and the other underwent a single sulfhydryl-disulfide exchange reaction, were generated during amylase denaturation by dodecyl sulfates.

Aspergillus oryzae↗
PubMed1995

Improved method for silver staining of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels.

A method for detection of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels was developed by a combination of (i) initial periodic acid oxidation/Alcian blue staining and (ii) subsequent staining with silver nitrate. The procedure allowed detection of as little as 1.6 ng of alpha 1-acid glycoprotein and 8-40 ng of a polydisperse mucin sample, which is at least 10 times more sensitive than previously published methods. The method should be very useful for assessment of sample purity and detection of glycoproteins in dilute mixed samples.

Alcian Blue↗
PubMed1995

Direct evaluation of stereoselectivity of cancer esterases by polyacrylamide gel electrophoresis coupled with activity staining with chiral naphthyl esters.

Both enantiomers of alpha-naphthyl 2-phenylpropanoate (PhPr(ONap)), N-acetylalaninate (AcAla(ONap)), N-methoxycarbonylalaninate (MocAla(ONap)), N-methoxycarbonylvalinate (MocVal(ONap)), N-acetylprolinate (AcPro(ONap)), and N-(trifluoroacetyl)prolinate (TfaPro(ONap)) were prepared and used with Fast Blue RR salt for activity staining of esterases separated by polyacrylamide gel electrophoresis. Comparison of the band thicknesses stained with each enantiomer indicates stereoselectivity of the major esterase in that band. Several esterases in normal rat liver, rat hepatoma-derived cells, and mouse B16 melanoma showed alteration or inversion of stereoselectivity by the substrate change from MocAla(ONap) to MocVal(ONap) or from AcPro(ONap) to TfaPro(ONap). The stereoselectivity of the major esterases for MocVal(ONap) was reversed between the murine liver and the B16 melanoma enzymes. The present staining with chiral naphthyl esters is very effective for surveying rapidly the stereoselectivity of animal tissue and cancer esterases.

Animals↗
PubMed1996

Membrane protein subunit fractionation by means of inverse pore gradient elution polyacrylamide gel electrophoresis.

We report here the preparative scale isolation of the four subunits of the nicotinic acetylcholine receptor (nAChR) applying short inverse pore gradient SDS gels on an elution-PAGE apparatus. The nAChR subunits are of similar molecular weights (alpha, 50.2 kDa; beta, 53.7 kDa; gamma, 56.3 kDa; delta, 57.6 kDa) and isoelectric point (approx 5.5) and share the typical properties of amphiphatic membrane proteins that are difficult to separate by chromatographic procedures. Preparative PAGE, which has proved to be the method of choice for nAChR-subunit fractionation, however, is time-consuming and achieves only moderate resolutions yielding dilute fractions. We present here the fractionation of a membrane preparation of Torpedo electric organ (2 mg of total protein) on short gels (2 cm) with linear or concave inverse pore gradients. All of the four subunits are isolated in homogeneous fractions of approx 70 micrograms/ml within only 4 h. Compared to the standard method, this means a 50% reduction in separation times with threefold higher concentrated protein fractions. We also give the theoretical explanation and experimental validation of the improved features resulting from short inverse-gradient polyacrylamide gels.

Animals↗
PubMed1996

Detection of protein kinase activities toward oligopeptides in sodium dodecyl sulfate-polyacrylamide gel.

In the previous paper, we reported a sensitive method for detection of protein kinase activities in gels after SDS-polyacrylamide gel electrophoresis (Kameshita, I., and Fujisawa, H. (1989) Anal. Biochem. 183, 139-143). This method is useful for the detection of various protein kinase activities toward protein substrates included in gels, but inapplicable to oligopeptide substrates because most of the oligopeptides eluted from the gel matrix during electrophoresis. The present study describes a new procedure for the detection of protein kinase activities toward synthetic oligopeptides in the gel. The oligopeptides which were linked to amino acid polymers such as poly-L-lysine through their amino-terminal cysteinyl residue by a heterobifunctional reagent were efficiently retained in the gel matrix and served as substrates for the protein kinases. As little as 2.5 pg of the catalytic subunit of cAMP-dependent protein kinase was detected by this in-gel assay method using a synthetic peptide as a substrate. This technique can be used for selective and sensitive detection of various protein kinases in crude tissue extracts.

Acrylic Resins↗
PubMed1996

An E-selectin binding assay based on a polyacrylamide-type glycoconjugate.

Here we show that biotinylated polyacrylamide-type glycoconjugates which contain sialyl Lewis X (sLex-polymer) or sialyl Lewis A (sLea-polymer) are ligands for E-selectin. sLea-polymer bound E-selectin with higher affinity than sLex-polymer. Based on this property we used the sLea-polymer to establish a sensitive cell-free binding assay for the characterization of E-selectin antagonists. The assay involves complexation of the biotinylated sLea-polymer with streptavidin-peroxidase. This complex is incubated with E-selectin mouse Ckappa fusion protein immobilized onto microtiter plates. Bound complex is detected by the peroxidase reaction. sLea-polymer bound in a Ca2+-dependent manner consistent with the function of E-selectin as a C-type lectin. Control glycoconjugates with sialic acid (alpha-Neu5Ac), Lewis A (Lea), or beta-D-glucose residues instead of sLea failed to interact with the E-selectin. Neutralizing anti-E-selectin antibodies blocked completely binding to E-selectin. This demonstrates specificity of the assay system. sLex blocked binding of the sLea-polymer to E-selectin by 50% at a concentration of 550 microM (IC50). The assay was used to characterize sLea-polymers with differing sLea content as multivalent inhibitors of E-selectin binding. The inhibitory activity of these polymeric forms of sLea increased with their sLea content up to IC50s in the low micromolar range. The binding assay described is sensitive, rapid, and simple and of low variability. Therefore it should be advantageous for the identification and characterization of novel E-selectin antagonists.

Acrylic Resins↗