LIT101C 9/1/98 9:39 AM Page Cvr1 Affi-Gel® Hz Immunoaffinity Kit Instruction Manual Catalog Number 153-6060 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
LIT101C 9/1/98 9:39 AM Page i Table of Contents Section 1 Introduction...............................................................1 1.1 Immunoaffinity Coupling and Usefulness of the Immobilized Antibody.........................................................1 1.2 Hydrazide Coupling Chemistry...........................................2 1.3 Diagram of Kit Components ...............................................2 Section 2 Monoclonal Antibodies.............................................
LIT101C 9/1/98 9:39 AM Page 1 Section 1 Introduction The Affi-Gel Hz immunoaffinity kit is a unique approach to IgG coupling to an agarose support matrix for affinity purification. This kit achieves a more uniform orientation of coupled antibody than currently available activated supports which couple via primary amines. Affi-Gel Hz hydrazide gel is an agarose support which reacts with the aldehydes of oxidized carbohydrates to form stable, covalent hydrazone bonds.
LIT101C 9/1/98 9:39 AM Page 2 1.2 Hydrazide Coupling Chemistry HOH2C HOH2C IgG IgG HOH2C IgG OH OH OH NalO4 OH O O Affi-Gel Hz gel OH N N NH NH Affi-Gel Hz gel Sugar residue of carbohydrate on the Fc region of IgG. Periodate oxidation of vicinal hydroxyls to form aldehydes. Oxidized IgG specific coupling to Affi-Gel Hz hydrazide gel. 1.
LIT101C 9/1/98 9:39 AM Page 3 Optional Bio-Rad Protein Assay Kit I (catalog number 500-0001) Section 2 Monoclonal Antibodies Due to the unique specificity of monoclonal antibodies, coupling to Affi-Gel Hz gel may not be optimal for all monoclonal antibodies. Loss of activity may occur depending on the individual monoclonal. To minimize loss of valuable antibody preparations, BioRad highly recommends that a sample coupling experiment be performed to determine the efficiency of the coupling reaction.
LIT101C 9/1/98 9:40 AM Page 4 matography may also be used to remove most serum components except transferrin. IgG in ascites fluid and serum may be purified by Affi-Gel protein A agarose (MAPS® II kit) low pressure chromatography, or by application to Affi-Prep® protein A medium to high pressure polymeric matrix. Protein A, a surface protein from Staphylococcus aureus, binds the Fc region of many mammalian IgG species. Protein A purification will yield highly purified IgG for coupling to Affi-Gel Hz gel.
LIT101C 9/1/98 9:40 AM Page 5 in affinity chromatography. 4.1A Dilution of Affi-Gel Hz 10x Coupling Buffer 1. Dilute Affi-Gel Hz 10x coupling buffer 1:10 with distilled, deionized water and mix well. 2. Check the pH of the diluted coupling buffer with a pH meter. The pH of the diluted buffer should be 5.5. If it is necessary to correct the pH of the diluted Affi-Gel Hz coupling buffer, use 1.0 M acetic acid or 1.0 M NaOH to bring the pH to 5.5. Sodium azide, at a concentration of 0.
LIT101C 9/1/98 9:40 AM Page 6 6. The Econo-Pac 10DG column should be washed with at least 20 ml of diluted coupling buffer, pH 5.5, to regenerate for desalting use after oxidation. 7. Place the yellow end cap over the column tip snugly to prevent leakage. Add 5 ml of diluted coupling buffer with 0.02% sodium azide to the column and replace the top cap for column storage. 8. The antibody is now ready for sodium periodate oxidation. 4.
LIT101C 9/1/98 9:40 AM Page 7 6. Immediately proceed to desalting, Section 4.3. 4.3 Desalting Procedure Immediately after the 1 hour oxidation, it is necessary to remove the sodium periodate from the IgG solution. Sodium periodate remaining in the IgG sample will adversely affect coupling efficiency. This desalting procedure is the same as that for buffer exchange (Section 4.1).
LIT101C 9/1/98 9:40 AM Page 8 4. Transfer the gel buffer slurry to a coupling reaction tube. If less than 5 ml of gel is to be used for coupling, wash only the volume of gel to be coupled. Unused gel should remain in isopropanol and be stored at 4 °C. 4.4B IgG Coupling As previously recommended, the IgG concentration should be between 1-5 mg/ml of gel. The total IgG sample volume limitation of 5 ml is suggested to facilitate buffer exchange and desalting in the Econo-Pac 10DG columns.
LIT101C 9/1/98 9:40 AM Page 9 Abs. @ 280 nm = (mg IgG/ml) x dilution factor x sample volume = total IgG 1.4 [total protein before coupling] - [total uncoupled protein (eluant + 0.5 M NaCl wash)] = [total coupled protein] (total coupled protein) x 100 = % protein coupled (total protein before coupling) The starting and final IgG solutions and 0.
LIT101C 9/1/98 9:40 AM Page 10 5.2 Sample Application 1. Sample is applied to the immobilized IgG column. Samples should be free of particulates. Complex samples should be diluted in application buffer and filtered if necessary. This will enhance specific binding to the immobilized IgG and prolong column life. 2. Wash column with 2 bed volumes of 0.5 M NaCl in application buffer to remove any unbound protein. 3. Wash column with 1-2 bed volumes of application buffer of lower NaCl concentration.
LIT101C 9/1/98 9:40 AM Page 11 Chaotropic Elution 4 M NaSCN 6 M urea 5 M guanidine-HC1 Elution Strategies The preceding eluants generally do not denature antibodies.* Eluant must be compatible with the antigen. Several methods should be tried in the following order: 1. Acid (pH 2-3.5) is common. May cause inactivity of some proteins. Can reduce solubility of IgG. 2. Chaotropic salts (3.5 M NaSCN, 3-6 M GuHCl) are often effective. Usually used at neutral pH. SCN should not be used with low pH.
LIT101C 9/1/98 9:40 AM Page 12 Catalog Number 130-0150 Product Description Bio-Gel HT Hydroxyapatite, 250 ml 156-0030 Macro-Prep High S Support, 100ml 156-0090 Macro-Prep t-Butyl HIC Support, 100ml 157-0040 Macro-Prep Ceramic Hydroxyapatite, type I, 100g 153-7304 CM Affi-Gel Blue Gel, 100ml 12
LIT101C 9/1/98 9:39 AM Page Cvr2 Bio-Rad Laboratories Life Science Group U.S.
