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DEAE column: FPLC

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.