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MOLECULAR BIOLOGY: WORKING WITH DNA

ANTIBODIES: ANTIGEN PREPARATION

Coupling of Cysteine-Containing Peptides to Epoxy-Activated Sepharose CL-6B Resin

Coupling of Cysteine-Containing Peptides to Epoxy-Activated Sepharose CL-6B Resin Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

Procedure Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. Weigh out 5 mg of the peptide containing an N- or C-terminal cysteine residue. 2. Put the peptide in a 15 ml conical tube and add 2.4 ml of ddH2O and 1.6 ml of 0.25 M Sodium Bicarbonate Buffer, pH 10, final concentration is 0.1 M NaHCO3 (see Hint #2). 3. Remove 4 μl for a small peptide gel and mix with 5 μl of 2X peptide gel sample Buffer. 4. Weigh out 0.4 g of Epoxy-Activated Sepharose CL-6B and place it in a 15 ml conical tube. 5. Add 10 ml of ddH2O to the dry resin and rotate the tube at room temperature for 15 min to allow the resin to swell. 6. Centrifuge the resin in a tabletop Beckman GS6R Centrifuge at 2500 rpm for 3 minutes. 7. Aspirate the supernatant and wash the resin twice more by resuspending it in 10 ml of ddH2O and recentrifugation. 8. Aspirate the final wash and leave the resin pellet. 9. Wash the resin once in 0.1 M Sodium Bicarbonate Buffer. 10. Centrifuge the resin in a tabletop Beckman GS6R Centrifuge at 2500 rpm for 3 minutes. 11. Aspirate the supernatant and leave the resin pellet. 12. Add the peptide solution to the resin and rotate the tube for 24 hr at room temperature to couple the peptide to the resin (see hint #3). 13. Add 0.1 volume (0.5 ml) of 1.0 M Ethanolamine HCl, pH 8.0 and rotate the resin for 5 to 6 hr at room temperature. 14. Centrifuge in a Beckman GS6R Centrifuge for 3 min at 2500 rpm. 15. Recover the supernatant to a new tube. 16. Mix 5 μl of the supernatant with 2X Peptide Gel Sample Buffer. 17. Wash the resin with Sodium Acetate/Chloride Solution. 18. Centrifuge in a Beckman GS6R Centrifuge for 3 min at 2500 rpm and remove the supernatant. 19. Wash the resin in Sodium Borate/Chloride Solution, centrifuge it in a Beckman GS6R Centrifuge for 3 min at 2500 rpm and aspirate the supernatant. Repeat this washing step twice. 20. Wash the resin 3 times with 10 ml of TEN and centrifuge in a Beckman GS6R Centrifuge for 3 min at 2500 rpm. 21. Remove the supernatant and store resin in an equal volume of TEN at 4°C. 22. Run a small peptide gel (Tris-Tricine; see Protocol for running Peptide Gels) to assess the coupling efficiency (see Hint #4).

Solutions Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

Peptide sample Buffer (2X)    2% (v/v) 2-mercaptoethanol or 3.1% (w/v) DTT 20% (v/v) glycerol 125 mM Tris 0.001% (w/v) Bromophenol Blue 4% (w/v) SDS TEN    0.02% (w/v) Sodium Azide (NaN3; CAUTION! see Hint #1) 10 mM Tris, pH 8.0 1 mM EDTA 1 M NaCl Sodium Borate/Chloride Solution    0.1 M Sodium Borate (Na2B4O7) pH 8.0 0.1 M NaCl Sodium Acetate/Chloride Solution    pH 4.0 0.1 M NaCl 0.1 M Sodium Acetate 1 M Ethanolamine HCl, pH 8.0    bring the volume to 100 ml with ddH2O pH to 8.0 with concentrated HCl 6.1 ml of Ethanolamine Free Base 0.1 M Sodium Bicarbonate Buffer, pH 10    22.5 ml of 1 M NaHCO3 27.5 ml of 1 M Na2HCO3 Bring volume to 200 ml with ddH2O 0.25 M Sodium Bicarbonate Buffer, pH 10    56.25 ml of 1 M Sodium Bicarbonate (NaHCO3) 68.75 ml of 1 M Sodium Carbonate (Na2HCO3) Bring volume to 200 ml with ddH2O 1.0 M NaOH

BioReagents and Chemicals Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

SDS EDTA Sodium Chloride N-methyl Pyrollidinone Sepharose CL-6B, Epoxy-Activated Ethanolamine, Free Base Sodium Acetate 2-Mercaptoethanol Sodium Hydroxide Sodium Borate Glycerol Sodium Azide Hydrochloric Acid Tris Sodium Carbonate Sodium Bicarbonate Dimethyl Formamide (DMF) Bromophenol Blue DTT

Protocol Hints Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. You can use Dimethyl Formamide (DMF; CAUTION! see Hint #1) or 30% N-methyl Pyrollidinone to help solubilize the peptide, if necessary. 3. Check the pH after 6 to 8 hr and add 1.0 M NaOH to keep the pH at 10. 4. Determine the coupling efficiency by measuring the amount of peptide present in the supernatant before and after coupling.