DEAE column: FPLC

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.