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Using Immunoprecipitation and 2DGE for Targeted Research into Protein Pathways
There are a variety of ways of using IP in conjunction with 2DGE. The following methods were developed for IP columns but can be adapted to a microfuge tube approach. Start by immunoprecipitating your proteins using your standard method. METHOD A:
- Wash beads/column extensively (10 column volumes) with TBS plus 0.02% Tween-20 (TBS-T). TBS alone can be used for a less stringent wash. If you suspect you are working with membrane proteins, the Tween can be replaced with Tris-buffered saline plus 0.1% Triton X-100 (TBS-TX).
- Elute proteins with four single column volumes of 0.1 M citric acid, pH 2.2 directly into a small volume (~1/10 volume) of 2M Tris pH 10 to neutralize. Mix the tubes immediately. The proteins primarily elute in the second and third fractions.
- If re-using the column, wash the column extensively with TBS-TX to neutralize.
Note: Instead of the low pH elution method, a high pH elution method is sometimes used with 50 mM diethylamine, pH 11.5, 0.1% Triton X-100 directly into four tubes with 1/20th column volumes of 1M NaH2PO4 (monobasic sodium phosphate), pH 4.2 to neutralize.
- Add sample buffer to the eluate. Try to keep the volume of the eluates as low as possible so that the sample buffer is not too diluted. We recommend starting with the Chaotropic buffer or the Modified O'Farrell's. You could do three preparations at the same time and compare the methods.
METHOD B:
1. After IP, beads can be washed with TE (50mM Tris-HCl, pH 7.5, 10mM EDTA, 1%SDS), then the immunoprecipitated material can be eluted using this buffer at 65°C for 10 min.
2. After allowing the eluate to cool to room temperature, dilute eluate 1:4 in Chaotropic 2D sample buffer. Tris is compatible with 2DGE, but not in high concentrations and it increases focusing time. EDTA can sometimes be problematic and concentrations should be kept low.
METHOD C:
IP is often accomplished using a kit. In this situation it is necessary to remove the salts from the elution buffer. This is done easily with a spin column, dialysis, or with a Centricon concentrator.
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