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Biotinylation Reagent (biotinylation + reagent)
Selected AbstractsSolid-phase biotinylation of antibodies,JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2004Elizabeth Strachan Abstract Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide,PEO2 biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS,PEO4 biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2,M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin,alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100,,g). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels. Copyright © 2004 John Wiley & Sons, Ltd. [source] A novel reactive ester derivative of biotin with reduced membrane permeability for in vivo biotinylation experimentsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2010Verena Strassberger Abstract The in vivo perfusion of rodent models of disease with biotin derivatives and the subsequent comparative proteomic analysis of healthy and diseased tissues represent a promising methodology for the identification of vascular accessible biomarkers. A novel, triply charged biotinylation reagent, NHS-,-Ala-(L -Asp)3 -biotin, was synthesized and validated in terms of its applicability for in vivo protein biotinylation. Compared to sulfo-NHS-LC-biotin, NHS-,-Ala-(L -Asp)3 -biotin exhibited a reduced membrane permeability and a preferential labeling of proteins localized in compartments readily accessible in vivo from the vasculature. [source] Mutation in hotfoot-4J mice results in retention of ,2 glutamate receptors in EREUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2002Shinji Matsuda Abstract The orphan glutamate receptor ,2 is selectively expressed in Purkinje cells and plays a critical role in cerebellar function. Recently, the ataxia of hotfoot-4J (ho-4J) mice was shown to be caused by a 170,amino acid deletion in the N-terminal region of ,2 receptors. To understand ,2 receptor function, we characterized these mutant receptors (,2ho) in Purkinje cells. Immunohistochemical staining showed that ,2ho receptors of the ho-4J homozygotes were abundantly expressed but localized to the Purkinje cell soma; in wild-type mice, ,2 receptors were predominantly present at distal dendrites of Purkinje cells. In addition, ,2ho receptors of the ho-4J mice were sensitive to endoglycosidase H, a finding suggesting that ,2ho receptors were not transported beyond the endoplasmic reticulum (ER) or cis -Golgi apparatus. To gain further insights into the mechanisms of this phenomenon, we characterized ,2ho receptors in transfected HEK293 cells. ,2ho receptors expressed in HEK293 cells were also sensitive to endoglycosidase H. Immunohistochemical staining showed that ,2ho receptors colocalized with proteins retained in the ER. Furthermore, ,2ho receptors were not labelled by membrane-impermeable biotinylation reagents. Coimmunoprecipitation assays showed that the intermolecular interaction of ,2ho receptors was significantly weaker than those of wild-type ,2 receptors, a finding suggesting that the ho-4J region is involved in oligomerization of ,2 receptors. Thus, ,2ho receptors were retained in the ER, probably by the quality control mechanism that detects unstable oligomers. We conclude that the absence of ,2 receptors on the cell surface by failed transport from the ER of Purkinje cells causes ataxia. [source] Solid-phase biotinylation of antibodies,JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2004Elizabeth Strachan Abstract Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide,PEO2 biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS,PEO4 biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2,M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin,alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100,,g). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels. Copyright © 2004 John Wiley & Sons, Ltd. [source] | ||||||||||