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| This protocol describes how to couple a peptide to CH Sepharose 4B resin using 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDAC) as a coupler. This protocol can be used to generate an affinity column to purify antibodies. |
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1. Add 4 ml of 0.5 M NaCl to 0.5 g of 6-Aminohexanoic Acid Sepharose 4B (CH Sepharose 4B).
2. Allow the resin to swell for 2 hr at room temperature.
3. Pour the solution onto a small 40C (40 mm, Coarse) (60 ml) sintered glass filter (see Hint #2).
4. Rinse the resin with 200 ml of 0.5 M NaCl.
5. Rinse the resin with 100 ml of pH 4.5 ddH2O.
6. Transfer the resin to a 15 ml conical tube using a spatula.
7. Dissolve 10 mg of the peptide to be coupled via the amino-terminus in 3 ml of pH 4.5 ddH2O. Check the pH and add the peptide solution to the resin solution. Mix by end-over-end rotation at room temperature.
8. Dissolve 115 mg of 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDAC) in 1 ml of pH 4.5 ddH2O.
9. Use 1.0 M HCl to bring the solution to pH 4.5 and add the crosslinker to the resin/peptide solution.
10. Mix by end-over-end rotation for 1 hr, checking the pH every 15 min. Bring pH to 4.5 with 1.0 M NaOH if necessary.
11. Mix by end-over-end rotation overnight.
12. Check that the pH is 4.5 and then block the unreacted groups by adding one-tenth volume of 1.0 M Ethanolamine, pH 8.0 (to 0.1 M final concentration).
13. Mix by end-over-end rotation for 3 hr at room temperature.
14. Transfer to a 60 ml sintered glass funnel as before and rinse the resin with 50 ml of Sodium Acetate/Chloride Solution and then with 50 ml of Sodium Bicarbonate/Chloride Solution. Repeat this alternating resin rinse step 5 times.
15. Rinse the resin with 80 ml of pH 4.5 ddH2O and then rinse with 80 ml of TEN.
16. Transfer the resin to a 15 ml conical tube with a spatula and add an equal volume of TEN With Sodium Azide. Store at 4°C in a tube sealed with Parafilm.
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| 1 M HCl |
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| 0.5 M NaCl |
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| TEN With Sodium Azide |
| 10 mM Tris, pH 7.0
1.0 M NaCl
1 mM EDTA
0.02% (w/v) Sodium Azide (CAUTION! see Hint #1)
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| TEN |
| 10 mM Tris, pH 7.0
1.0 M NaCl
1 mM EDTA
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| Sodium Bicarbonate/Chloride Solution |
| 0.5 M NaCl
0.1 M NaHCO3
pH 10
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| Sodium Acetate/Chloride Solution |
| 0.5 M NaCl
pH 4.5
0.1 M Sodium Acetate
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| 1 M Ethanolamine Hydrochloride, pH 8.0 |
| Bring volume to 100 ml with ddH2O
pH to 8.0 with concentrated HCl
6.1 ml Ethanolamine Free Base (liquid)
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| 1 M NaOH |
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| pH 4.5 ddH2O |
| Adjust pH of ddH2O to 4.5 with HCl
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Sodium Azide
Hydrochloric Acid
EDTA
1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDAC)
Tris
Sodium Hydroxide
6-Aminohexanoic Acid Sepharose 4B (CH Sepharose 4B)
Sodium Chloride
Sodium Bicarbonate
Ethanolamine, free base (liquid)
Sodium Acetate
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. A sintered glass funnel is a glass funnel with a frit in it. The C stands for coarse. The frits come in grades: extra coarse, coarse, medium, fine, extra fine. The 40 stands for 40 mm.
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