INSTRUCTIONS Imject® Maleimide Activated cBSA 77155 77175 0869.2 Number Description 77155 Imject Maleimide Activated cBSA, 10 mg 77175 Imject Maleimide Activated cBSA, 2 mg Supplied: Lyophilized in PBS with EDTA, pH 7.2 with proprietary stabilizer Maleimide activation: ≥ 4 moles of maleimide/mole cBSA Storage: Upon receipt store product at 4°C. Product is shipped at ambient temperature. Introduction Imject Maleimide Activated cBSA is a carrier protein used for antibody production. The maleimide activation allows specific conjugation of sulfhydryl-containing molecules to elicit an immune response and antibody production against the conjugated molecule. The carrier is pre-activated using Sulfo-SMCC (Product No. 22322), a heterobifunctional cross-linker that contains a N-hydroxysuccinimide (NHS) ester and a maleimide group. The activated carrier presents an available maleimide group that can react with sulfhydryl-containing peptides or proteins. The cBSA is bovine serum albumin (BSA) that has been modified by substituting anionic carboxyl groups with cationic aminoethyl-amide groups. When used as an immunogen, this cationized BSA (cBSA) stimulates a much higher antibody response than native BSA. In vivo, antibody response is not only increased but remains elevated for an extended period. In vitro, much less cBSA than native BSA is required to produce the same degree of T cell proliferation. The underlying mechanism is related to the form of immunogen that is recognized by the cells regulating the response. Because of its net positive charge (pI > 11), cBSA has a greater affinity for the negatively charged cell surface membrane of the antigenpresenting cell. Rather than pinocytosis, internalization of cBSA occurs by receptor-mediated endocytosis, an adsorptive mechanism that results in more efficient uptake and processing of the immunogen. The cBSA is effective with both haptens and proteins for eliciting an enhanced immunological response toward the coupled molecule, which eliminates the need to develop protocols for each antigen system. This carrier will produce an anti-peptide response greater than a traditional carrier and is effective for enhancing the antibody response to proteins with low isoelectric points. Important Product Information • Maleimides react with sulfhydryls at pH 6.5-7.5 to form stable thioether bonds. At pH values > 7.5, reactivity toward primary amines and hydrolysis of the maleimide group can occur; however the maleimide group of Sulfo-SMCC is unusually stable up to pH 7.5. • Molecules for conjugation must have free sulfhydryl (–SH) group(s) available. Cysteine-containing molecules often oxidize in solution and form disulfide bonds, which cannot react with maleimides. Disulfide bonds can be reduced to produce free sulfhydryls. After reduction, most reducing reagents must be removed before conjugation. The ReduceImm™ Reducing Kit (Product No. 77700) and Immobilized TCEP Disulfide Reducing Gel (Product No. 77712) enable peptide or protein reduction while recovering the sample in the absence of reducing agents. • Sulfhydryls can be added to molecules using N-succinimidyl S-acetylthioacetate (SATA, Product No. 26102) or 2-iminothiolane•HCl (Traut’s Reagent, Product No. 26101). Both reagents modify primary amines. Pierce Biotechnology PO Box 117 (815) 968-0747 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax www.thermo.com/pierce Procedure for Hapten-Carrier Conjugation The protocol is designed to yield effective immunogens for a wide variety of haptens but is not necessarily optimal for a specific hapten. Differences in size and structure of haptens will affect conjugation efficiencies. Using a molar excess of hapten (peptide) over maleimide groups ensures complete and efficient conjugation. Generally, reacting equal mass amounts of hapten and cBSA will achieve sufficient molar excess. If a molar excess of hapten is not available, add a sulfhydrylcontaining compound, such as cysteine, after conjugation to quench any remaining active maleimide groups. A. Additional Materials Required • Conjugation Buffer: BupH™ Phosphate Buffered Saline Packs, each pack results in 0.1 M sodium phosphate, 0.15 M NaCl; pH 7.2 when reconstituted with 500 ml of ultrapure water (Product No. 28372), or other buffer at pH 6.5-7.5 • Desalting column (e.g., D-Salt™ Polyacrylamide Desalting Column, Product No. 43240) or dialysis cassettes (e.g., Slide-A-Lyzer® Dialysis Cassette Kit, Product No. 66382) • Imject® Purification Buffer Salts (Product No. 77159), contains 0.083 M sodium phosphate, 0.9 M NaCl and a proprietary stabilizer (see note below) Note: If the conjugate is to be used for injection within one week, use PBS for desalting. If the conjugate will be frozen, use the Purification Buffer Salts for desalting, which will preserve the product during freeze-thaw cycles. B. Conjugation Procedure 1. Reconstitute one vial of the maleimide-activated protein by adding ultrapure water to make a 10 mg/ml solution. 2. Dissolve the sulfhydryl-containing hapten in a volume of Conjugation Buffer equal to 1.0-2.5 times the volume of reconstituted activated protein. For example dissolve 2 mg of peptide in 200-500 µl of buffer for addition to 2 mg of activated carrier reconstituted in 200 μl water. Alternatively, if the peptide is freely soluble it may be added as a solid to the protein solution. Note: For haptens with limited solubility, DMSO may be used for solubilization. Use ≤ 30% DMSO in the final conjugation solution or the carrier protein may irreversibly denature. 3. Immediately mix the peptide and activated protein and react for 2 hours at room temperature. 4. Purify the conjugate by desalting or dialysis to remove EDTA, which should not be injected into laboratory animals. Additional Notes: • PBS may be used for conjugate purification. If the conjugate will be frozen, use the Purification Buffer Salts, which will preserve the product during freeze-thaw cycles. • Desalting or dialysis will not separate non-conjugated protein; however, a large excess of hapten is used in this protocol, making it unlikely that non-conjugated carrier exists in significant quantity. • If DMSO was used in the conjugation, add DMSO to the Purification Buffer Salts for desalting to prevent precipitation in the column; dialysis is not compatible with DMSO. • If a precipitate has formed during conjugation, centrifuge the material, collect the supernatant and save the precipitate. Use only the supernatant for purification. Combine the purified conjugate to the precipitate. • To purify antibodies specific to the peptide, prepare an affinity column by immobilizing the peptide through the same functional group used to prepare the immunogen. The SulfoLink® Coupling Gel (Product No. 20402) contains an activated gel support that will couple peptides via sulfhydryl groups. The peptide affinity column can then be used to specifically bind anti-peptide antibodies from serum, allowing antibodies against the carrier protein to flow through the column. Peptide-specific antibodies can then be eluted and recovered. Pierce Biotechnology PO Box 117 (815) 968-0747 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax 2 www.thermo.com/pierce Information Available from the Web Please visit the Pierce web site for additional information relating to this product including the following items: • Tech Tip Protocol: Remove air bubbles from columns • Tech Tip Protocol: Degas solutions for use in affinity columns • Tech Tip Protocol: Prepare a measurable and reversible sulfhydryl-reactive affinity column • Tech Tip: Protein stability and storage Related Pierce Products 77140 Imject® Freund’s Complete Adjuvant, 5 × 10 ml 77145 Imject® Freund’s Incomplete Adjuvant, 5 × 10 ml 77161 Imject® Alum, 50 ml 77138 AdjuPrime™ Immune Modulator, 20 mg 15150 Reacti-Bind™ Maleimide Activated Plates (8-well Strip), 5 plates 66382 Slide-A-Lyzer® Dialysis Cassette Kit, 10 dialysis cassettes, each appropriate for 0.5-3.0 ml samples 45212 Melon™ Gel IgG Purification Kit, sufficient reagents to purify IgG from up to 50 ml of serum 45206 Melon™ Gel IgG Spin Purification Kit, sufficient reagents to purify up to 3 ml of serum Cited Reference 1. Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 56-100. General References Muckerheide, A., et al. (1987). Cationization of protein antigens. I. Alteration of immunogenic properties. J. Immunol. 138:833-7. Muckerheide, A., et al. (1987). Cationization of protein antigens. II. Alteration of regulatory properties. J. Immunol. 138:2800-4. Domen, P.L., et al. (1987). Cationization of protein antigens. III. Abrogation of Oral Tolerance. J. Immunol. 139: 3195-8. Apple, R.J., et al. (1988). Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells. J. Immunol. 140:3290-5. The cBSA is protected by US Patent 5,142,027. The most current versions of all product instructions are available at www.piercenet.com. For a faxed copy, contact customer service (in the USA call 800-874-3723) or your local distributor. This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”). No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s). There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii) use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products. Current versions of product instructions are available at www.thermo.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor. © 2008 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA. Pierce Biotechnology PO Box 117 (815) 968-0747 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax 3 www.thermo.com/pierce