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Handbook & Selection Guide

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Handbook & Selection Guide Antibody Production Handbook & Selection Guide c (#JPTDJFODFTttXXX(#JPTDJFODFTDPN t Protein Estimation Assays t Lysis Bu!ers & Systems t Apoptosis Assays t Protein Fractionation Kits t Cytotoxicity Assays t Dialysis (Micro) System t SAM Methyltransferase Assays t Electrophoresis Clean-Up t Protease Assays t Concentration Systems t Phosphatase Assays t Contamination Removal t Peroxide Assay t Protease Inhibitor Cocktails t Proteomic Grade Detergents t Individual Protease Inhibitors t Research Grade Detergents t Protease Assays t Non-Ionic, Ionic & Zwitterionic t Proteases for Mass Spec. t Detergent Estimations t Sequencing Grade Proteases t Detergent Removal Systems t 1-Hour Western System t Gel Preparation Chemicals t Transfer Bu!ers & Membranes t Protein Marker Ladders t Membrane Stains t Electrophoresis Bu!ers t Blocking Bu!ers t Reducing & Alkylating Reagents t Secondary Antibodies t Protein Gel Stains t Detection Reagents t Reprobing Reagents t Protein Sample Preparation t A"nity Resins t Protein Clean-Up Systems t 6X His Protein Puri#cation Kits t Electrophoresis Reagents t GST Protein Puri#cation Kits t Mass Spec Grade Protease t Antibody Puri#cation t InGel Digestion Kits t Activated Resins t Peptide Generation Reagents t Bu!ers & Reagents t Biotin Labeling t Carrier Proteins t Cell Surface Protein Labeling t Peptide Coupling Systems t Agarose Coupling Kits t Antibody Puri#cation Resins t Fluorescent Dye Labeling Kits t Antibody Fragmentation Kits t Enzyme Labeling Systems t Coated Plates t Blocking Bu!ers t Homobifunctional t Wash Bu!ers t Heterobifunctional t Secondary Antibodies t Optimizer Systems t Detection Reagents t Cross-Linking Systems t Antibody Labeling Systems t Apoptosis Assays t DNA Isolation t Cytotoxicity Assays t Transformation & Screening t SAM Methyltransferase Assays t Polymerase Chain Reaction t Protease Assays t Agarose Electrophoresis t Phosphatase Assays t RNA Isolation t Peroxide Assay t Yeast Transformation t ELISA Table of Contents Introduction 2 Antibody Fragmentation 13 The Immune Response........................................................................2 Immobilized Papain..............................................................................13 Immobilized Pepsin ..............................................................................13 Antibody Production 3 Immobilized Ficin ..................................................................................13 Carrier Proteins Fab Fragmentation ...............................................................................13 KLH .............................................................................................................3 F(ab)2 Fragmentation ...........................................................................14 ..................................14 Bovine Serum Albumin (BSA) ...........................................................3 Fab & F(ab)2 Fragmentation of Mouse IgG1 HyperCarrier™ ........................................................................................4 Antibody Labeling 15 Activated Carrier Proteins4 ActiveHOOK™ Carrier Proteins ..........................................................4 Fluorescent Dye Labeling5 HOOK™ Dye Labeling Kit (5/6) TAMRA-SE (Rhodamine) .........15 Peptide Coupling Kits5 HOOK™ Dye Labeling Kit (FITC) ........................................................15 HOOK™ Peptide Coupling (Amine Reactive) ...............................5 OneQuant™ Fluorescent Reagents .................................................15 HOOK™ Peptide Coupling (Sulfhydryl Reactive) ........................5 Biotin Labeling Antibody Puri!cation 6 Biotin Selection Guide .........................................................................17 OneQuant™ Biotin Reagents .............................................................19 Protein A & Protein G HOOK™ Biotin Kits .................................................................................19 Immobilized Protein A .........................................................................6 Micro HOOK™ Biotin Kits ....................................................................20 Immobilized Protein G ........................................................................6 HOOK™ BiotinQuant .............................................................................20 Pearl™ Puri!cation HOOK™ IgG Biotinylation ...................................................................20 Pearl™ IgG Puri!cation Resin .............................................................7 HOOK™ BiotinQuant .............................................................................21 Pearl™ IgG Puri!cation Kits ................................................................7 Avidin .........................................................................................................21 Pearl™ Monoclonal IgG Puri!cation Kit .........................................7 HABA ..........................................................................................................21 Pearl™ Antibody Clean Up Kit ...........................................................8 Streptavidin Resin .................................................................................21 IgA Puri!cation Avidin Resin .............................................................................................22 Immobilized Jacalin ..............................................................................8 Monomeric Avidin Resin.....................................................................22 Jacalin, Lyophilized ...............................................................................8 &O[ZNF-BCFMJOH Thiophilic Adsorption HOOK™ HRP PLUS Labeling ...............................................................23 Thiophilic Resin ......................................................................................8 HOOK™ HRP Sulfo Labeling ...............................................................23 HOOK™ AP Sulfo Labeling ..................................................................23 "óOJUZ$PMVNO(FOFSBUJPO9 Sulfhydryl Coupling Resin ..................................................................9 Antibody Puri!cation Accessories 23 Sulfhydryl Immobilization Kit for Proteins ...................................9 Sulfhydryl Immobilization Kit for Peptides ..................................9 %JTQPTBCMF$PMVNOT Amine Coupling Resin .........................................................................10 Spin Column, <0.1ml............................................................................23 CDI Amine Reactive Resin ..................................................................10 Spin Column, 1ml ..................................................................................23 Carboxyl Coupling Resin ....................................................................11 Spin Column, 2ml ..................................................................................24 SDC™ (Steroid/Drug/Compound) Immobilization ....................11 Spin Column, 3ml ..................................................................................24 $PBUFE8FMM1MBUFT Spin Column, 5ml ..................................................................................24 Spin Column, 10ml ...............................................................................24 Well-Coated™ Protein A, Protein G & Protein A/G......................12 Well-Coated™ Protein L .......................................................................12 Well-Coated™ Antibody ......................................................................12 Updated: Jul 28, 2011 For further details, visit www.GBiosciences.com 1 Introduction Antibodies, in particular the production of speci!c antibodies, in the acidi!ed endosomes and lysosomes, digest the complex into have been essential in the advancement of many scienti!c !elds, small peptides (4). particularly proteomics, immunology and cell biology. The immature MHC class II (MHCII) proteins wait for the detection A common practice for the generation of speci!c antibodies is the of foreign material in the endoplasmic reticulum, where it is in a use of immunogenic peptides derived from the protein of interest. complex with the Invariant chain (Ii) (5). Upon detection of the foreign These peptides are short sequences of amino acids that are antigenic, material, the MHCII:Ii complex leaves the ER, traverses the Golgi however they lack elements required for T cell activation. Activation and endosomes and !nally fuses with the MHCII compartment (6). is achieved with the use of “carrier proteins” that are coupled to the Cathepsins, in the MHCII compartment, partially digest Ii chain leaving small antigenic peptide. CLIP in the peptide-binding site. The endosome/lysosome, with the carrier protein:peptide The Immune Response peptides, fuses with the MHCII compartment and a human leukocyte The coupling of the peptide to a carrier protein is important in the antigen (HLA) protein replaces CLIP with a carrier protein:peptide stimulation of the immune response and the generation of antibodies. peptide (7). The mature, peptide-loaded MHC class II protein moves Below is a brief description and diagram summarizing the immune to the plasma membrane and is presented on the cell surface (8). pathway responsible for detecting the peptide and carrier protein and A speci!c helper T-cell binds the peptide-loaded MHC class generating subsequent antibodies. II protein through its T-cell receptor and CD4 receptor (9), which
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