The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Richard R. Burgess - One of the best experts on this subject based on the ideXlab platform.
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Identification of polyol-responsive monoclonal antibodies for use in Immunoaffinity Chromatography.
Current protocols in molecular biology, 2020Co-Authors: Nancy E. Thompson, Richard R. BurgessAbstract:One of the limitations of Immunoaffinity Chromatography as been that high-affinity antigen-antibody complexes are difficult to dissociate, often leading to inactivation of the protein product during elution from the immobilized antibody. As described in this unit, some antigen-antibody complexes can be dissociated in the presence of a combination of a low-molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt. These conditions seem to be generally nondenaturing and, in some cases, even protein-stabilizing. This type of antibody is designated "polyol-responsive." These antibodies can be easily identified and isolated as monoclonal antibodies (MAbs) from a typical fusion, using standard hybridoma procedures. They have proven to be very valuable reagents for the Immunoaffinity purification of active, labile, multi-subunit protein complexes.
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Identification, production, and use of polyol-responsive monoclonal antibodies for Immunoaffinity Chromatography.
Methods in Enzymology, 2009Co-Authors: Nancy E. Thompson, Katherine M. Foley, Elizabeth S. Stalder, Richard R. BurgessAbstract:Abstract Immunoaffinity Chromatography is a powerful tool for purification of proteins and protein complexes. The availability of monoclonal antibodies (mAbs) has revolutionized the field of Immunoaffinity Chromatography by providing a continuous supply of highly uniform antibody. Before the availability of mAbs, the recovery of the target protein from immobilized polyclonal antibodies usually required very harsh, often denaturing conditions. Although harsh conditions are often still used to disrupt the antigen–antibody interaction when using a mAb, various methods have been developed to exploit the uniformity of the antigen–antibody reaction in order to identify agents or conditions that gently disrupt this interaction and thus result in higher recovery of active protein from Immunoaffinity Chromatography. We discuss here the use of a specific type of monoclonal antibody that we have designated “polyol-responsive monoclonal antibodies” (PR-mAbs). These are naturally occurring mAbs that have high affinity for the antigen under binding conditions, but have low affinity in the presence of a combination of low molecular weight hydroxylated compounds (polyols) and nonchaotropic salts. Therefore, these PR-mAbs can be used for gentle Immunoaffinity Chromatography. PR-mAbs can be easily identified and adapted to a powerful protein purification method for a target protein.
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Development of an epitope tag for the gentle purification of proteins by Immunoaffinity Chromatography: application to epitope-tagged green fluorescent protein.
Analytical Biochemistry, 2003Co-Authors: Nancy E. Thompson, Terrance M. Arthur, Richard R. BurgessAbstract:Abstract Polyol-responsive monoclonal antibodies (mAbs) are useful tools for the gentle purification of proteins and protein complexes. These are high-affinity mAbs that release the antigen in the presence of a nonchaotropic salt and a low-molecular-weight polyhydroxylated compound (polyol). The epitope for the polyol-responsive mAb NT73, which reacts with Escherichia coli RNA polymerase, was located at the C terminus of the β′ subunit. Using recombinant DNA techniques, we have identified the epitope to be within the 13-amino-acid sequence SLAELLNAGLGGS and have developed an epitope tag that can be fused to a protein of interest for use as a purification tag. This epitope tag (designated Softag1) was fused to either the N or the C terminus of the green fluorescent protein. These tagged proteins were expressed in E. coli, and the tagged proteins were purified from the soluble fraction by a single-step Immunoaffinity Chromatography procedure. This approach extends the powerful technique of gentle-release Immunoaffinity Chromatography to many expressed proteins.
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advances in gentle Immunoaffinity Chromatography
Current Opinion in Biotechnology, 2002Co-Authors: Richard R. Burgess, Nancy E. ThompsonAbstract:Abstract Immunoaffinity Chromatography is one of the most powerful fractionation steps available for protein purification; however, it is often difficult to elute bound protein without using harsh or denaturing elution conditions. The development of methods to identify monoclonal antibodies that bind antigens tightly, but release under gentle, non-denaturing conditions has made possible the Immunoaffinity purification of labile, multisubunit enzyme complexes with high yield and high specific activity. This work has implications for emerging proteomic applications, allowing identification of new protein–protein interaction partners, retention of biological activity and the isolation of protein complexes more amenable to crystallization and structure determination.
Nancy E. Thompson - One of the best experts on this subject based on the ideXlab platform.
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Identification of polyol-responsive monoclonal antibodies for use in Immunoaffinity Chromatography.
Current protocols in molecular biology, 2020Co-Authors: Nancy E. Thompson, Richard R. BurgessAbstract:One of the limitations of Immunoaffinity Chromatography as been that high-affinity antigen-antibody complexes are difficult to dissociate, often leading to inactivation of the protein product during elution from the immobilized antibody. As described in this unit, some antigen-antibody complexes can be dissociated in the presence of a combination of a low-molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt. These conditions seem to be generally nondenaturing and, in some cases, even protein-stabilizing. This type of antibody is designated "polyol-responsive." These antibodies can be easily identified and isolated as monoclonal antibodies (MAbs) from a typical fusion, using standard hybridoma procedures. They have proven to be very valuable reagents for the Immunoaffinity purification of active, labile, multi-subunit protein complexes.
-
Identification, production, and use of polyol-responsive monoclonal antibodies for Immunoaffinity Chromatography.
Methods in Enzymology, 2009Co-Authors: Nancy E. Thompson, Katherine M. Foley, Elizabeth S. Stalder, Richard R. BurgessAbstract:Abstract Immunoaffinity Chromatography is a powerful tool for purification of proteins and protein complexes. The availability of monoclonal antibodies (mAbs) has revolutionized the field of Immunoaffinity Chromatography by providing a continuous supply of highly uniform antibody. Before the availability of mAbs, the recovery of the target protein from immobilized polyclonal antibodies usually required very harsh, often denaturing conditions. Although harsh conditions are often still used to disrupt the antigen–antibody interaction when using a mAb, various methods have been developed to exploit the uniformity of the antigen–antibody reaction in order to identify agents or conditions that gently disrupt this interaction and thus result in higher recovery of active protein from Immunoaffinity Chromatography. We discuss here the use of a specific type of monoclonal antibody that we have designated “polyol-responsive monoclonal antibodies” (PR-mAbs). These are naturally occurring mAbs that have high affinity for the antigen under binding conditions, but have low affinity in the presence of a combination of low molecular weight hydroxylated compounds (polyols) and nonchaotropic salts. Therefore, these PR-mAbs can be used for gentle Immunoaffinity Chromatography. PR-mAbs can be easily identified and adapted to a powerful protein purification method for a target protein.
-
Development of an epitope tag for the gentle purification of proteins by Immunoaffinity Chromatography: application to epitope-tagged green fluorescent protein.
Analytical Biochemistry, 2003Co-Authors: Nancy E. Thompson, Terrance M. Arthur, Richard R. BurgessAbstract:Abstract Polyol-responsive monoclonal antibodies (mAbs) are useful tools for the gentle purification of proteins and protein complexes. These are high-affinity mAbs that release the antigen in the presence of a nonchaotropic salt and a low-molecular-weight polyhydroxylated compound (polyol). The epitope for the polyol-responsive mAb NT73, which reacts with Escherichia coli RNA polymerase, was located at the C terminus of the β′ subunit. Using recombinant DNA techniques, we have identified the epitope to be within the 13-amino-acid sequence SLAELLNAGLGGS and have developed an epitope tag that can be fused to a protein of interest for use as a purification tag. This epitope tag (designated Softag1) was fused to either the N or the C terminus of the green fluorescent protein. These tagged proteins were expressed in E. coli, and the tagged proteins were purified from the soluble fraction by a single-step Immunoaffinity Chromatography procedure. This approach extends the powerful technique of gentle-release Immunoaffinity Chromatography to many expressed proteins.
-
advances in gentle Immunoaffinity Chromatography
Current Opinion in Biotechnology, 2002Co-Authors: Richard R. Burgess, Nancy E. ThompsonAbstract:Abstract Immunoaffinity Chromatography is one of the most powerful fractionation steps available for protein purification; however, it is often difficult to elute bound protein without using harsh or denaturing elution conditions. The development of methods to identify monoclonal antibodies that bind antigens tightly, but release under gentle, non-denaturing conditions has made possible the Immunoaffinity purification of labile, multisubunit enzyme complexes with high yield and high specific activity. This work has implications for emerging proteomic applications, allowing identification of new protein–protein interaction partners, retention of biological activity and the isolation of protein complexes more amenable to crystallization and structure determination.
Beata Halassy - One of the best experts on this subject based on the ideXlab platform.
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nonspecific native elution of proteins and mumps virus in Immunoaffinity Chromatography
Journal of Chromatography A, 2016Co-Authors: Marija Brgles, Dora Sviben, Dubravko Forcic, Beata HalassyAbstract:Immunoaffinity Chromatography, based on the antigen-antibody recognition, enables specific purification of any antigen (protein, virus) by its antibody. The problem with Immunoaffinity Chromatography is the harsh elution conditions required for disrupting strong antigen-antibody interactions, such as low pH buffers, which are often deleterious for the immobilized protein and the protein to be isolated since they can also disrupt the intramolecular forces. Therefore, Immunoaffinity Chromatography can only be partially used for protein and virus purification. Here we report on a nonspecific elution in Immunoaffinity Chromatography using native conditions by elution with amino acid solution at physiological pH for which we suppose possible competing mechanism of action. Elution potential of various amino acid solutions was tested using Immunoaffinity columns specific for ovalbumin and mumps virus, and protein G affinity column. Results have shown that the most successful elution solutions were those containing imidazole and arginine of high molarity. Imidazole represents aromatic residues readily found at the antigen-antibody interaction surface and arginine is most frequently found on protein surface in general. Therefore, results on their eluting power in Immunoaffinity Chromatography, which increases with increasing molarity, are in line with the competing mechanism of action. Virus Immunoaffinity Chromatography resulted in removal on nonviable virus particles, which is important for research and biotechnology purposes. In addition, amino acids are proven stabilizers for proteins and viruses making approach presented in this work a very convenient purification method.
Yonekazu Hamano - One of the best experts on this subject based on the ideXlab platform.
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application of Immunoaffinity Chromatography for detection of tetrodotoxin from urine samples of poisoned patients
Toxicon, 1999Co-Authors: Kentaro Kawatsu, Tadayoshi Shibata, Yonekazu HamanoAbstract:Abstract Immunoaffinity Chromatography using the monoclonal antibody (T1-1) specific for tetrodotoxin (TTX) has been developed for isolating TTX from urine samples. By combining Immunoaffinity Chromatography with fluorometric high performance liquid Chromatography (HPLC), it has become possible to detect a small amount of TTX in urine samples. The detection limit of TTX in urine was 2 ng/ml. By this combined method, TTX was detected in all the urine samples that were collected from poisoned patients during the week following TTX ingestion. The combination of Immunoaffinity Chromatography with HPLC was very useful in detecting TTX from the urine samples of poisoned patients for diagnosis of TTX-food poisoning.
Marija Brgles - One of the best experts on this subject based on the ideXlab platform.
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nonspecific native elution of proteins and mumps virus in Immunoaffinity Chromatography
Journal of Chromatography A, 2016Co-Authors: Marija Brgles, Dora Sviben, Dubravko Forcic, Beata HalassyAbstract:Immunoaffinity Chromatography, based on the antigen-antibody recognition, enables specific purification of any antigen (protein, virus) by its antibody. The problem with Immunoaffinity Chromatography is the harsh elution conditions required for disrupting strong antigen-antibody interactions, such as low pH buffers, which are often deleterious for the immobilized protein and the protein to be isolated since they can also disrupt the intramolecular forces. Therefore, Immunoaffinity Chromatography can only be partially used for protein and virus purification. Here we report on a nonspecific elution in Immunoaffinity Chromatography using native conditions by elution with amino acid solution at physiological pH for which we suppose possible competing mechanism of action. Elution potential of various amino acid solutions was tested using Immunoaffinity columns specific for ovalbumin and mumps virus, and protein G affinity column. Results have shown that the most successful elution solutions were those containing imidazole and arginine of high molarity. Imidazole represents aromatic residues readily found at the antigen-antibody interaction surface and arginine is most frequently found on protein surface in general. Therefore, results on their eluting power in Immunoaffinity Chromatography, which increases with increasing molarity, are in line with the competing mechanism of action. Virus Immunoaffinity Chromatography resulted in removal on nonviable virus particles, which is important for research and biotechnology purposes. In addition, amino acids are proven stabilizers for proteins and viruses making approach presented in this work a very convenient purification method.
