Purification of cAR1 from CHIFF

1. CHIFF was thawed and solubilized in 1-2 lubrol-PX (substitute 0.1% sarkosyl or 0.1% DM since lubrol-PX is no longer available) in TBP for 2-4 hrs with mixing at 4^o at a cell equivalent density of 3-4 x 10⁸ /ml.

2. After centrifugation at 100,000g for 30 min, the supernatant was recovered. NaCl and immidazole were added at final concentrations of 200 mM and 2 mM, respectively. About 40 ml of this supernatant was first batch-incubated with 1.0 ml (sedimented volume) of metal-chelating sepharose resin (Pharmacia) precharged with 50 mM Ni⁺⁺ according to manufacturer’s suggestion.

3. After 2-4 hrs incubation, the resin was spun down and loaded onto a 5 ml Bio-Rad econocolumn. The supernatant was further absorbed over the column by gravity-flow (for about 1 hr).

4. Wash the column with more than 20 ml of wash buffer ( 40 mM Tris-HCl, pH7.5, 200 mM NaCl, 2 mM immidazole, and 0.1% lubrol), the column was further washed with buffer containing 15 mM nonylglucoside (40 mM Tris, 200 mM NaCl, 2 mM imidazole and 15 mM nonylglucoside) to exchange the original lubrol. This was found to be necessary since our result showed that NG preserved receptor binding activity better than lubrol.

5. cAR1 was eluted in four steps of increasing concentrations of immidazole: 25 mM, 50 mM, 100 mM amd 250 mM. Each step consisted of four column-volumes of elution solution.

6. cAR1 usually started to come out at second 50 mM fraction wash and peaked between 100-250 mM elution.

 

This page was last edited 06/26/2003