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1. Dialyze the protein sample into Coupling Buffer to get rid of any SDS or Tris buffer. SDS and Tris may inhibit the coupling reaction. The protein concentration should be 0.5 to 1 mg for 3 to 5 ml of buffer.
2. Save a 50 μl aliquot of the protein sample in Coupling Buffer for later analysis of coupling efficiency on a SDS polyacrylamide minigel (see Protocol on SDS-Polyacrylamide Mini-Gels).
3. Allow the Reacti-Gel agarose beads to warm up to room temperature.
4. Take 2 to 4 ml of the agarose bead slurry for a 1 ml column of settled beads to a 15 ml, 20c glass-fritted funnel with gentle suction. Do not let the beads dry out.
5. Wash with 3 volumes of ice-cold ddH2O, followed by 3 volumes of ice-cold Coupling Buffer.
6. Take 1 ml of settled, wet, and washed agarose beads and place them in a 15 ml conical tube using a spatula to make the transfer. Rinse the filter with Coupling Buffer to transfer all of the beads to the tube.
7. Remove the excess Coupling Buffer from the agarose beads with a Pasteur pipette.
8. Add 750 μg of protein sample per ml of agarose beads (Add protein in 3 to 5 ml Coupling Buffer).
9. Place on a tube inverter and incubate at 4°C overnight.
10. Let the beads settle and remove a 50 μl aliquot of coupling reaction supernatant and save it for comparison with the pre-coupling protein concentration on a SDS polyacrylamide mini-gel.
11. Add 0.1 volume (i.e. 100 μl for a 1 ml bead volume) 1 M Ethanolamine-HCl, pH 8.0.
12. Incubate for 3 hr at room temperature on a tube inverter.
13. Put the beads into a 15 ml 20c glass-fritted funnel with gentle suction. Do not let the beads dry out. Run 6 column volumes of 1 M NaCl though the funnel.
14. Run 6 column volumes of ice-cold ddH2O though the funnel.
15. Run 6 column volumes of Glycine/NaCl though the funnel.
16. Re-equilibrate the beads with PBS until the pH of the solution reaches 7.0 (Check the pH with pH paper).
17. Place the PBS-equilibrated beads into a 15 ml conical tube.
18. Add sodium azide to 0.02% (w/v) and store at 4°C (see Hint #1).
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| 1 M NaCl |
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| Glycine/NaCl |
| 150 mM NaCl
pH 2.5
50 mM Glycine
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| 1 M Ethanolamine-HCl, pH 8.0 |
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| Coupling Buffer |
| Store at 4°C
Adjust pH to 8.5 with 4 M NaOH
0.1 M Sodium Borate
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| 4 M NaOH |
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| PBS |
| pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (NaH2PO4)
137 mM NaCl
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Sodium Azide
Glycine
Ethanolamine-HCl
Sodium Hydroxide
Sodium Borate
Potassium Chloride
Sodium Chloride
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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