Phosphocellulose (Whatman P-11) Resin

The phosphocellulose P-11 resin is sold by Whatman (catalog #4071-010 PDF) and frequently used to purify DNA-binding proteins. The resin is sold as a dry powder and needs to be properly hydrated before it can be used. The protocol below was kindly provided by Dr. Karen Browning (dead link).

Contents

1. Washing procedure
2. Preparation of PC column

1. Washing procedure

1. Place ~300 ml of dry resin in a 2 liter flask. Fill flask with deionized water and suspend the resin by swirling and/or inverting the flask. Allow resin to settle for about 30 min. Decant the water, discarding as much of the fines as possible. Repeat washing 8-10 times, discarding the fines each time. It is very important to remove the fines from Phosphocellulose! If the fines are not removed, the column will run very slowly.
2. Suspend resin in 1 liter of 0.2 M NaOH. (Do this in a hood because sometimes ammonia will be released.) Allow to stand for at least 30 min. Decant NaOH, add another liter of 0.2 M NaOH and check pH of supernatant with pH indicator paper. The pH should be above 10; if not, wash with more NaOH.
3. Suspend resin in deionized water and wash with deionized water until the pH of the wash is below 8.
4. Suspend resin in 1 liter of 0.2 M HCl. Allow to stand for at least 30 min. Decant HCl, add another liter of 0.2 M HCl and check pH of supernatant with indicator paper. The pH should be below 3; if not, wash with more HCl.
5. Suspend resin in deionized water and wash with deionized water until pH of wash is about 5 (or pH of water).
6. Suspend resin in 500 ml of 1 M Tris/HCl, pH 7.6. Allow to stand at least 6 h or overnight. Decant buffer, add another 500 ml of 1 M Tris buffer and check pH. The pH should be 7.6; if not, wash with 0.5 Tris buffer until pH is 7.6.
7. Suspend resin in 0.5 M Tris/HCl and store at 4°C for as long as 1 week. If stored longer, add 0.02 % sodium azide. Fresh buffer should be added about every two weeks as ammonia will continue to leach off the resin.

2. Preparation of PC column

1. To prepare an ~8 ml column, place about 10 ml of resin in a 50 ml flask, decant 0.5 M Tris and wash resin twice with ~10 ml of portions of deionized water.
2. Wash twice with ~10 ml portions of desired buffer.
3. Pour about 10 ml buffer into column (1-1.5 cm diameter) and check flow. Pour suspended resin into column and allow resin to pack.
4. Wash with about 20 ml of buffer at a flow rate of 20 ml/hr.
   Check pH of buffer coming out of column; be sure it is 7.6!

After column has been used, the resin can be regenerated once; same as described for washing.