Bio-Rad Affi-Gel 15 Gel - Manual (Page 19)

hydrophobic interactions. Chaotropic anions are
effective in the order SCN->ClO-
4
>I>Br->Cl-. 6,7
Chaotropic cations are effective in the order
guanidine>Mg
2+ >K + >Na + . 7 Eluants such as 8 M urea,
6 M guanidine hydrochloride, and 6 M NaSCN are
effective in disrupting most protein-protein interac-
tions. The problem is that these strong chaotropes
may destroy the activity of the antigen and/or the anti-
body. Conditions as mild as possible should always
be used.
When the eluant has been chosen, the elution condi-
tions should be refined by optimizing concentration, time,
temperature, and by combining the eluants described
above.
It is important to remove the eluted antigen or anti-
body from the eluant as quickly as possible to minimize
the chance of denaturation. If acid or base is used, the
33
3. Base Elution is less frequently used than acid elution,
but, in some cases, it is more effective. Elution with
1 M NH
4
OH or with 50 mM diethylamine, pH 11.5,
has been shown to be effective with membrane glyco-
proteins and with certain antigens which precipitate in
acid but are stable in base.
3 Organic solvents can also
be added to basic eluants as described above with acid
elution. An example in which an antibody to dinitro-
phenyl-bovine serum albumin (DNP-BSA) was cou-
pled to an affinity matrix is described in Reference 5.
DNP-BSA could not be eluted at all with acid or with
acid plus organic solvents. Base elution gave 60%
yield and base plus dioxane gave 95% yield of puri-
fied antigen.
4. Chaotropic Agents disrupt the tertiary structure of
proteins and, therefore, can be used to dissociate anti-
gen-antibody complexes. Chaotropic salts disrupt
ionic interactions, hydrogen bonding, and sometimes
32
LIT156B 6/17/98 10:41 AM Page 32