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At least 667 records · Page 37Linked to original sources
PubMed1984

"Golden blot"--detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes.

Protein A bound to particles of colloidal gold is widely used for cytochemical localization of antigens. A method for detecting immune complexes on "Western blots" with protein A-gold has been developed. It involved the following steps: (i) electrophoretic separation of proteins, (ii) transfer to nitrocellulose, (iii) incubation with antibodies, and (iv) staining with protein A-gold. The amount of antigen detectable was 10-50 ng. The sensitivity could be improved by subsequent photochemical silver staining (1-10 ng detected), and was then comparable to autoradiograms using iodinated protein A.

Animals↗
PubMed1984

Immunogold for detection of antigen on nitrocellulose paper.

The application of immunogold for the detection of antigen-antibody complexes on nitrocellulose paper is described. Tobacco mosaic virus (TMV) protein, either purified or in leaf extract, was bound to the nitrocellulose paper and then exposed to rabbit anti-TMV serum. The antigen-antibody complexes were detected by gold-labeled goat anti-rabbit immunoglobulin G. The gold-labeled antibody is directly visible because of its pink color. This method can detect 1-5 pg of TMV protein, either in purified form or in the unpurified plant extract, with high specificity.

Animals↗
PubMed1984

An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gels.

A new apparatus for electrophoretic transfer of proteins from micro polyacrylamide slab gels has been developed. The apparatus enabled the easy changing of nitrocellulose sheets and was suited for obtaining multiple blots from a gel. Electrophoretic conditions were determined so that all of the blots obtained sequentially from one slab gel were successfully used to visualize specific proteins irrespective of their molecular weight. Combining the transfer technique with the technique of parallel micro two-dimensional electrophoresis, 20 blots could be obtained within 1 h of electroblotting time. The locations of 28 human serum proteins were determined simultaneously on these blots using commercial specific antisera.

Blood Proteins↗
PubMed1984

A simple assay of solubilized adenosine receptors with nitrocellulose membrane filters.

A rapid and simple assay of solubilized adenosine receptors with nitrocellulose membrane filters is described. This assay was sensitive and reproducible when applied to adenosine receptors solubilized from rat brainstem membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Appropriate values of dissociation constants for the solubilized adenosine receptors to their tritiated agonists were obtained by the membrane filter technique. This method should be applicable for the assay of a variety of solubilized receptors.

Adenosine↗
PubMed1984

Immunoblot method for identifying surface components, determining their cross-reactivity, and investigating cell topology: results with Haemophilus influenzae type b.

Methods currently used for identifying exposed membrane components of gram-negative bacteria can give false positive results, and also do not provide information on nonprotein components such as lipopolysaccharide and polysaccharide capsules. A method, described within, has been developed to overcome these limitations. Briefly an outer membrane preparation derived from encapsulated (type b) Haemophilus influenzae was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated components were then transferred electrophoretically to nitrocellulose. The nitrocellulose was cut into vertical strips, which were then each incubated with rabbit antiserum to the whole bacterium or with the same antiserum after absorption with any of the following: the same strain of H. influenzae, a capsule-deficient mutant of that strain, other strains of H. influenzae, or other bacteria. The strips were then incubated with 125I-protein A, and the bound antibodies were detected by autoradiography. The autoradiograph of the strip exposed to unabsorbed antisera revealed the identity of those individual outer membrane components that bound antibodies. A comparison of the intensity of the various bands on this strip with those on the strips exposed to absorbed antisera was then used to identify (1) surface-exposed components, (2) those components occluded by capsule, and (3) cross-reactivity of exposed components. This method should be applicable to other cells and subcellular particles. Its major disadvantage is that it can provide false negative results.

Autoradiography↗
PubMed1984

Calibration of prestained protein molecular weight standards for use in the "Western" or enzyme-linked immunoelectrotransfer blot techniques.

Prestained protein molecular weight standards allow easy, direct visual location of electrophoretically transblotted lanes on nitrocellulose. They also provide a simple and accurate means for calibrating the molecular weights of resolved bands. Commercial prestained protein molecular weight standards, however, appear to have significantly different molecular weights from the original unstained proteins. We describe a calibration of these prestained molecular weight standards.

Collodion↗
PubMed1984

Monoclonal antibody screening: two methods using antigens immobilized on nitrocellulose.

The dot-immunobinding assay of Hawkes et al. [R. Hawkes, E. Niday, and J. Gordon (1982) Anal. Biochem. 119, 142-147] has been modified such that many antibody solutions are screened simultaneously on dots of antigen applied to a single sheet of nitrocellulose using very small amounts of both antigen and antibody solutions. This method is also used in the assay of antibody binding on dots of components of the antigen after enzymatic digestion and chromatographic fractionation and to dots of compounds which may be chemically related to the antigen. An aluminum template for screening Western blots with a number of different antibody solutions on a single sheet of nitrocellulose is also described. Possible applications of the dot-immunobinding assay in screening for virus are discussed.

Animals↗
PubMed1985

Properties and applications of new monoclonal antibodies raised against calf DNA polymerase alpha.

A panel of 12 hybridoma cell lines secreting monoclonal antibodies against alpha-polymerase were prepared by fusion of mouse myeloma cells and spleen cells of a rat immunized with homogeneous calf thymus alpha-polymerase. Hybridomas were selected and cloned on the basis of immunobinding to pure alpha-polymerase in solid phase radioimmunoassay. Antibodies secreted by these cells eventually were purified in milligram quantities from ascites fluids. These antibodies, all of the rat immunoglobulin M class, cross-reacted with alpha-polymerases from calf and monkey cells as revealed by immunobinding in radioimmunoassay and by immunoprecipitation of DNA polymerase activity. The antibodies were not capable of neutralizing the enzyme activity. With the methods described these antibodies may be used to immunoprecipitate alpha-polymerase from crude extracts of mammalian cells and to measure levels of the enzyme protein.

Animals↗
PubMed1985

Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents.

A rapid and convenient method was established for analysis of the N-linked carbohydrate chains of glycoproteins on nitrocellulose sheets. Proteins were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, reacted with peroxidase-coupled lectins, and detected by color development of the enzyme reaction. Four glycoproteins having N-linked oligosaccharide chains were used as test materials: Taka-amylase A (which has a high-mannose-type chain), ovalbumin (high-mannose-type chains and hybrid-type chains), transferrin (biantennary chains of complex type), and fetuin (triantennary chains of complex type and O-linked-type chains). Concanavalin A interacted with Taka-amylase A, transferrin, and ovalbumin but barely interacted with fetuin. After treatment of the glycoproteins on a nitrocellulose sheet with endo-beta-N-acetylglucosaminidase H, transferrin reacted with concanavalin A but Taka-amylase A and ovalbumin did not. Wheat germ agglutinin interacted with Taka-amylase A but not ovalbumin; therefore, they were distinguishable from each other. Fetuin and transferrin were detected by Ricinus communis agglutinin or peanut agglutinin after removal of sialic acid by treatment with neuraminidase or by weak-acid hydrolysis. Erythroagglutinating Phaseolus vulgaris agglutinin detected fetuin and transferrin. Thus, the combined use of these procedures distinguished the four different types of N-linked glycoproteins. This method was also applied to the analysis of membrane glycoproteins from sheep red blood cells. The terminally positioned sugars of sialic acid, alpha-fucose, alpha-galactose, and alpha-N-acetylgalactosamine were also detected with lectins from Limulus polyphemus, Lotus tetragonolobus, Maclura pomifera, and Dolichos biflorus, respectively.

Binding Sites↗
PubMed1985

Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface-conductive glass as anode.

A new type of inexpensive horizontal apparatus for the electrophoretic transfer of proteins from a gel to an immobilization membrane has been developed. In this system, gel and membrane were directly pressed between two flat electrodes. A surface-conductive glass was used as anode and a stainless-steel plate as cathode. Proteins could be transferred from polyacrylamide gel to nitrocellulose sheet, with a yield of at least 90% in 60-90 min, without overheating, using a voltage gradient of 30-40 V/cm. Moderate volumes of separate anodic (with 20% methanol) and cathodic (with 0.1% sodium dodecyl sulfate) buffers were suited for optimal transfer of proteins with relative molecular mass (Mr) in the 14,000-94,000 range.

Animals↗
PubMed1985

A direct antigen-binding assay to screen hybridoma supernatants.

A simple and convenient method of directly assaying hybridoma supernatants for the desired monoclonal antibodies is described which obviates the need for labeled second or third antibody conjugates. Culture supernatants (1-5 microliters) were directly spotted onto a nitrocellulose sheet, and additional protein binding sites blocked with bovine serum albumin and incubated with enzyme-labeled, radioactive, or fluorescent antigen. Positive hybridoma supernatants were identified after washing and detection of bound antigen by appropriate means.

Antibodies, Monoclonal↗
PubMed1985

Protein blotting in uniform or gradient electric fields.

Experimental data and computer mapping were used to analyze electric fields generated by a variety of electrode arrays in protein blotting apparatus. Asymmetric electrode arrays were found to generate nonuniform fields that effected uneven transfers of 125I-labeled albumin from gels to nitrocellulose membranes. Symmetric arrays with multiple (four), independent wire electrodes, supplied individually with electric current, generated the most uniform fields and effected the most even transfers of the test protein. With multiple independent electrodes, gradient electric fields can be generated in which differences in electrophoretic elution between large and small proteins can be eliminated. Transfer apparatus with either uniform or gradient electric fields are expected to improve qualitative results and make possible quantitation of protein blotting.

Collodion↗
PubMed1985

Immunoblot specificity of monoclonal antibodies assayed against complex extracts.

Monoclonal antibodies of known specificity, and target cells of known antigen expression, were used to evaluate the specificity of the immunoblot assay against complex extracts. The antibody panel included 10 previously characterized monoclonals to antigens of known structure: HLA-A,B,C; Ia; and actin. In addition, a commercial reagent was shown to react with HLA chains in immunoblots, and not to cross-react with actin. Target cells known to have strong, weak, or negative expression of the antigens were examined. In all cases, the results were in complete accord with the established properties of the antibodies, antigens, and target cells. For example, HLA bands were detected in lymphoid cells with strong HLA-A,B,C expression, and also in a neuronal cell line with 0.5% of the lymphoid activity. Cytoskeletal cross-reactions, or other unexpected specific bands, were not observed. This work lays a foundation for interpreting immunoblot analyses of monoclonal antibodies to newly defined proteins of complex tissue.

Actins↗
PubMed1985

Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes.

Methods to detect "native" proteins immobilized on nitrocellulose membranes in spot tests or on blots prepared from polyacrylamide slab gels after electrophoretic separation are described. Gold sols were found to be useful as general stains for proteins: They are polychromatic, yield an indelible record, and are complementary to india ink as protein stains because these two stains have different sensitivities for a number of proteins tested. For detection of wheat germ lectin (WGL)-binding glycoproteins, avidin-peroxidase was an effective enzyme probe, because the glycoportion of the avidin moiety possesses binding affinity to WGL. Glycocomponents in human parotid saliva were detected with this probe and with the following biotin-conjugated lectins as intermediary probes: soybean lectin, Bandeiraea simplicifolia lectin, Lotus tetragonolobus lectin, and kidney bean lectin. Autoclaving blots prior to probing eliminated endogenous peroxidase activity. Concanavalin A and WGL were separated by isoelectric focusing and detected on blots with horseradish peroxidase and avidin-peroxidase, respectively. The versatility of the biotin/avidin system was used to detect other lectins on similar blots using biotin-conjugated glycoproteins as intermediary probes: Helix pomatia lectin and B. simplicifolia lectin were detected with biotinyl neoglycoproteins, and kidney bean lectin with biotin-conjugated components of parotid saliva.

Biotin↗
PubMed1985

In situ detection of beta-lactamase activity in sodium dodecyl sulfate-polyacrylamide gels.

After beta-lactamase had been denatured by boiling in the presence of sodium dodecyl sulfate (SDS) and then electrophoresed in SDS-polyacrylamide gels, activity could be restored and could be detected in situ as specific molecular species. Renaturation was simple and facilitated by the presence of a carrier protein. The assay was sensitive, detecting 0.8 ng beta-lactamase activity in the gel.

Bacillus↗
PubMed1985

Use of different hapten-protein conjugates immobilized on nitrocellulose to screen monoclonal antibodies to abscisic acid.

The dot-immunobinding method for screening antibodies to proteins on sheets of nitrocellulose has been modified to allow monoclonal antibodies (McAb) to the hapten abscisic acid (ABA) to be screened. Several methods for conjugating ABA to proteins using new bifunctional coupling reagents, specific for hapten keto groups, are described. Hybridomas secreting McAb with a defined specificity for the hapten can be identified by screening supernatants against the carrier protein and other hapten-protein conjugates with different conjugation bridges or modified hapten structure. Inhibition of binding to conjugates by free hapten is used to determine the relative avidity of the McAb for free and bound hapten. All of these tests could be done with no more than about 50 microliter of antibody solution. Dot immunobinding is a useful alternative to radioimmunoassay for screening McAb to haptens.

Abscisic Acid↗
PubMed1985

Protein analyses and reagents: microscale assay of calcium-binding activity of proteins and peptides using a nitrocellulose membrane.

A simple and reliable method for the measurement of calcium binding to proteins and peptides was developed. It is composed of two procedures--filtration through a nitrocellulose membrane filter and estimation of 45Ca retained on the membrane. The routine assay was completed within a few minutes, and only microgram amounts of samples were necessary. This method permitted the quantitative determination of the calcium-binding activity of proteins and peptides including one with a molecular weight of as low as 4000. This method also permitted the detection of low-affinity interactions (Kd congruent to 10(-3) M), possibly because the nitrocellulose membrane did not show nonspecific binding of calcium and because a washing step was not employed in the routine assay. Ultrafiltration membranes when used in the apparatus gave no useful data.

Animals↗