Contributor: Suprya Jayadev
Date: September 21,1993
Resin preparation:
1)Measure out appropriate volume of resin into a 50 ml conical tube.
--> NOTE: 1 ml of DEAE-Sephacel should bind approximately 100 mg BSA.
2)Remove ethanol from resin by washing resin 5X with a maximal volume of ice-cold water each time.
3)Wash resin one time with 1M Tris,pH 7.6.
4)Wash resin 2 times with lysis buffer containing no DMSO or ATP.
--> Equilibrate resin with buffer for 5 minutes each time.
5)Check the pH of the resin to make sure it is at or near pH 7.5.
6)Add sample to resin and equilibrate (with continual mixing)for 1 to 2 hours.
Column preparation and run:
7)Pack resin in column,draining diluent constantly.
--> MAKE SURE that no bubbles form in the column matrix!!
8)Wash column with 5 column volumes of lysis buffer containing no DMSO!!
--> NOTE: DMSO strips proteins off of the coulumn!
9)Run a 40 ml salt gradient collecting 1 to 2 ml fractions:
- Start at 0 mM NaCl and end with 500 mM NaCl.
10)Assay protein peaks for SMase activity.
