Coupling Cysteine Containing Peptides to Carrier Protein for Immunization and Polyclonal Antibody

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Coupling Cysteine Containing Peptides to Carrier Protein for Immunization and Polyclonal Antibody

Date Added: April 11, 2008 09:30:46 PM

Author: plinkadmin

Category: Peptide Protocols

The peptide sequence must contain Cysteine amino acids in order for the crosslinking agent to efficiently couple the peptides to the carrier protein (RSA). Once coupled, the peptide-carrier protein conjugate can be used in a schedule of injections to elicit an immune response in rabbits against the peptide sequence. 1. Prepare Sephadex G-25 fine beads by swelling them overnight in 0.1 M NaH2PO4, pH 6.0 at room temperature (5 ml buffer/dry gram of beads). 2. Wash the resin 4 times using 0.1 M NaH2PO4, pH 6.0. 3. Pour Sephadex G-25 into a 130 mm x 20 mm column or equivalent. 4. Wash with 15 ml of 0.1 M NaH2PO4, pH 6.0 to equilibrate the column and to settle the resin into the column. 5. Add the SMCC drop-wise to the RSA solution with mixing (see Hint # 2 and #4). 6. Incubate the mixture at room temperature on a rotating mixer for 30 min. 7. Add 200 μl of Dimethylformamide (DMF) to 1 ml of 0.1 M NaH2PO4, pH 6.0 8. Dissolve 5 mg of the Cysteine-containing peptide in 0.1 M NaH2PO4, pH 6.0/DMF solution form Step #7. 9. Incubate at room temperature until ready to couple. 10. Add 1 ml of 0.1 M NaH2PO4, pH 6.0 to the SMCC-RSA conjugate and mix well. 11. Load the conjugate onto the Sephadex G-25 column. 12. Use 0.1 M NaH2PO4, pH 6.0 as the column buffer and gravity flow to collect 1 ml fractions in 1.5 ml tubes. 13. Collect 12 fractions total (see Hint #5). 14. Assay each fraction by Microtiter Plate Bradford Assay for protein using approximately 2 μl of each fraction (see Protocol on Protein Concentration Determination Using Microtiter Plate Bradford Assay). 15. Pool the peak fractions of SMCC-RSA conjugate into a 15 ml conical tube. 16. Add the dissolved peptide solution (from Step #9) to the tube. 17. Incubate the tube for 12 to 16 hr at room temperature on a rotating mixing wheel (see Hint #6). 18. Dialyze the SMCC-RSA-peptide conjugate for 24 hr against 1 liter of PBS with 2 changes of buffer at 4�C. Use 3,500 molecular weight cut off dialysis tubing.(see Hint #7). 19. Divide the sample into 400 μl aliquots and store at -80�C. 20. To produce antibodies, use 100 μl of the carrier-crosslinker-peptide conjugate per rabbit injection every three weeks with Freund's Adjuvant (see Protocol for Preparation of Polyclonal Antibodies). 0.1 M NaH2PO4, pH 6.0 �� 0.1 M Sodium Phosphate Monobasic, pH 6.0 PBS (10X) �� 1.4 M NaCl pH 7.2 30 mM KCl 20 mM Potassium Phosphate Monobasic (KH2PO4) 40 mM Sodium Phosphate Dibasic (Na2HPO4) MBS �� Prepared in 150 μl of Dimethylformamide (DMF) Optional: either prepare SMCC OR MBS (See Hint #2) 1 mg m-Maleimidobenzoyl-N-Hydroxysuccinimide ester (MBS)(Pierce) SMCC �� Prepared in 150 μl of Dimethylformamide (DMF) (CAUTION see Hint #3) 1 mg Succinlmidyl 4-[n-Maleimidomethyl-Cyclohexane-1-Carboxylate (SMCC) (Pierce) Optional: either prepare SMCC OR MBS (See Hint #2) Keyhole Limpet Hemocyanin (KLH) �� 4 mg Keyhole Limpet Hemocyanin Optional: either prepare RSA OR KLH (See Hint #1) Dissolved in 0.3 ml of 0.1 M NaH2PO4, pH 6.0 Dialyze overnight against 0.1 M NaH2PO4, pH 6.0 Rabbit Serum Albumin (RSA) �� Optional: either prepare RSA OR KLH (See Hint #1) Dissolved in 0.3 ml of 0.1 M NaH2PO4, pH 6.0 Dialyze overnight against 0.1 M NaH2PO4, pH 6.0 4 mg Rabbit Serum Albumin Potassium Phosphate, Monobasic Sodium Phosphate, Dibasic Freund's Adjuvant Potassium Chloride Sodium Chloride m-Maleimidobenzoyl-N-Hydroxysuccinimide Ester Keyhole Limpet Hemocyanin Succinlmidyl 4-[n-Maleimidomethyl-Cyclohexane-1-Carboxylate Rabbit Serum Albumin Sodium Phosphate Monobasic Dimethylf ...

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