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Excess Reagent (excess + 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] Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MSELECTROPHORESIS, Issue 8 2010Zoltan Szabo Abstract CE is a high-resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ,100 fold excess of fluorophore reagent, 8-aminopyrene-1,3,6-trisulfonic-acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:,100 ratio) maintained the >95% derivatization yield at 55°C with only 50,min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8-aminopyrene-1,3,6-trisulfonic-acid simplified the removal of excess reagent, important in both CE-LIF (electrokinetic injection bias) and CE-MS (ion suppression). Coupling CE- ESI-MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values. [source] Determination of bisphenol A in rat brain by microdialysis and column switching high-performance liquid chromatography with fluorescence detectionBIOMEDICAL CHROMATOGRAPHY, Issue 5 2002Yen Sun A sensitive column switching HPLC-fluorescence detection for determination of bisphenol A (BPA) in rat brain by coupling with microdialysis was developed. A microdialysis probe was inserted into the hypothalamus of rat brain and an artificial cerebrospinal fluid was used for perfusion. BPA in brain dialysate was subjected to a fluorescent derivatization with 4-(4,5-diphenyl-1H -imidazol-2-yl)benzoyl chloride (DIB-Cl), and the excess reagent was removed by a column-switching technique. Separation was carried out on two ODS semimicro-columns with the mobile phase of acetonitrile,H2O,methanol,tetrahydrofuran (55:10:35:2.5, v/v) and acetonitrile,0.1,M acetate buffer (pH 3.0),methanol (35:10:55, v/v) at a flow rate of 0.10 and 0.15,mL/min for a precolumn and a separation column, respectively. Fluorescence intensity was monitored at 475,nm with excitation of 350,nm. BPA could be sensitively detected at 0.3 ppb in 60,µL brain microdialysate at a signal-to-noise ratio of 3. By the proposed method, concentrations of BPA in rat brain and plasma were monitored for 8,h after single i.v. or oral administration. It is proved that BPA is capable of penetrating the blood,brain barrier. The ratio of the area under the concentration,time curve of BPA in rat brain to that in blood was estimated to be about 3.0,3.8%. Copyright © 2002 John Wiley & Sons, Ltd. [source] Thiopropyl-agarose as a solid phase reducing agent for chemical modification of IgG and F(ab,)2BIOTECHNOLOGY PROGRESS, Issue 5 2008Natalia Ferraz Abstract Selective reduction of native disulfide bonds in immunoglobulins is one of the best methods for introducing reactive groups on to the protein surface. Additionally, the thiol groups so generated may allow oriented conjugation at a specific site of the immunoglobulin. Solid-phase reducing agents have many advantages over soluble ones (including ease of separation of excess reagent from reduced protein by filtration, and the potential for regeneration and multiple reuse). In this work we report a comparative study of the reduction of rabbit IgG and its F(ab,)2 fragments, with mercaptohydroxypropylether-agarose (thiopropyl-agarose), a solid phase reducing agent, and dithiothreitol. The effect of different parameters on the process, such as the amount of reducing agent, incubation period, and temperature, was assessed by titration of thiol groups and SDS-PAGE analysis. Optimized reduction with thiopropyl-agarose introduced six thiol groups in the F(ab,)2 fragment (mol/mol). Native IgG was less reactive, probably due to steric effects, as only an average of three thiol groups were introduced. However, by increasing reaction temperature from 22 to 37°C, six thiol groups could be introduced in native IgG (mol/mol). Reduction with dithiothreitol also introduced six thiol groups in F(ab,)2 fragments (mol/mol) but led to higher thiol content for the whole IgG. These results demonstrated that thiopropyl-agarose can be a very useful tool for exercising more precise control over the reduction treatment, and for selecting which disulfide bridges are to be broken. After 6 h incubation with reducing agent containing 8 and 16 ,moles SH per mg of protein, the resulting reduced IgG retained the same biological activity as the native immunoglobulin. The controlled modification of native disulfides achieved with thiopropyl-agarose will be useful for the development of soluble and insoluble immunoglobulin conjugates. [source] Preparation of kinase-biased compounds in the search for lead inhibitors of kinase targets,MEDICINAL RESEARCH REVIEWS, Issue 3 2005Justine Y.Q. Lai Abstract This work describes the preparation of approximately 13,000 compounds for rapid identification of hits in high-throughput screening (HTS). These compounds were designed as potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds, e.g., purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the hydrogen bond acceptor/donor properties known to be important for kinase binding. Several of these are based upon literature kinase templates, or adaptations of them to provide novelty. The routes to their preparation are outlined. A variety of automation techniques were used to prepare >500 compounds per scaffold. Where applicable, scavenger resins were employed to remove excess reagents and when necessary, preparative high performance liquid chromatography (HPLC) was used for purification. These compounds were screened against an ,in-house' kinase panel. The success rate in HTS was significantly higher than the corporate compound collection. © 2004 Wiley Periodicals, Inc. [source] | |||||||||||||