Bio-Rad Affi-Gel Blue Gel - Manual (Page 14)

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Suggested Procedure for Enzyme Purification
1. Prepare a column of Affi-Gel blue gel, 100-200 mesh.
A 5 ml bed volume for every 20 mg of protein to be
chromatographed should be sufficient. Equilibrate the
column with starting buffer. The starting buffer should
be of low ionic strength, 50 mM or less. (Published
methods have used pH values from 6.0 to 8.5)
2. Dialyze sample against starting buffer. Alternatively,
the sample can be rapidly desalted in a column of
Bio-Gel P-6 DG gel, on an Econo-Pac 10DG
column, or on an Econo-Pac P6 cartridge.
3. Apply the sample to the column.
4. Wash the column with 2 bed volumes of starting
buffer.
5. Check the effluent for enzyme activity. If it is not
bound, then alter conditions. Change the pH,
decrease the ionic strength, or change the buffer.
6. The column may be eluted with a salt gradient or
with a competitive eluant such as a cofactor. Table 2
contains examples of eluants used in Affi-Gel blue
gel chromatography.
LIT590B 9/3/98 1:30 PM Page 12