Bio-Rad Affi-Gel 15 Gel - Manual (Page 12)

5.2 Anhydrous Coupling
Coupling under anhydrous condition is the preferred
method when the solubility of the ligand permits. It is
ideal for peptides. Since there is no hydrolysis of active
esters in the absence of water, the only reaction which will
take place is the one between the ester and the ligand.
Material and equipment required for anhydrous cou-
pling using Affi-Gel 10 and Affi-Gel 15 gel.
1. Cold (anhydrous) isopropanol.
2. Organic solvent of your choice; alcohol, dimethylsul-
foxide (DMSO), dioxane, formamide free from free
amines, or mixtures of these solvents.
3. Closed container which holds four times the volume
of gel to be used.
4. (Optional) rotating shaker.
5. (Optional) Buchner funnel.
19
solution per ml of gel, and agitate sufficiently to make a
uniform suspension. Continue gentle agitation of the gel
slurry on a rocker, shaker, or wheel for 1 hour at room
temperature or 4 hours at 4 °C.
If the coupling time is short, or if the gel is to be used
immediately, we recommend a precautionary blocking of
any active esters. This can be accomplished by adding 0.1 ml
1 M glycine ethyl ester (pH 8) or 0.1 ml 1 M ethanolamine
HCl (pH 8) per ml gel. Allow 1 hour for completion of the
blocking reaction. Transfer the gel to a column and wash
with water or coupling buffer until the gel is free of reac-
tants, as detected by O.D.
280
. Wash the gel with other sol-
vents that will be used subsequently to elute substances
specifically bound to the column. The column is now ready
for equilibration with starting buffer and application of sam-
ple. When not in use, store the columns at 4 °C, and in a
starting buffer containing 0.2% sodium azide.
18
LIT156B 6/17/98 10:41 AM Page 18