DEAE Column

Contributor: Suprya Jayadev

Date: April 11,1996

1)Weigh out the appropriate amount of DEAE sephadex and place in a 15 ml conical tube.

2)Wash resin three to four times with zenopure water.Mix gel with water by repeated inversion,then centrifuge at 1,000 - 2,000 rpm;40℃

3)Wash two times with 100 mM Tris,pH 7.5.

- Equilibrate 5 minutes with gentle inversion prior to spinning down the resin.

4)Equilibrate resin with the starting buffer (sb)for a period of 5 mins.

- Starting buffer is the buffer in which the sample is in.Any salt that is in the sb.

5)Check the pH of the equilibrated mix.It should be approximately 7.56,if not adjust accordingly.

6)Spin gel down again and add sample to the resin.

7)Allow sample to equilibrate with the resin by mixing on the rotary shaker (in cold room)at a speed of 3 for a period of 40 mins.

8)Load gel onto column and collect flow through.

9)Wash column with 5 column volumes of starting buffer.

- In actuality,a more rigorous manner in which to check washing is to monitor A280.Once absorbance has leveled off at zero,the column is sufficiently washed.

10)Elute column with the appropriate salt solutions.

- When eluting SMase activity,elute:

a)three times with (1 ml of)100 mM NaCl.

b)two times with (0.7 ml)of 250 mM NaCl.