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        DEAE column: FPLC

        互联网

        1721

        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.

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