Bio-Rad Profinity Epoxide Resin - Manual (Page 10)

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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, is
effective with membrane glycoproteins and certain antigens that precipitate in acid
but are stable in base (Izuta and Saneyoshi 1988). Organic solvents can also be
added to basic eluants as described above with acid elution.
Chaotropic agents disrupt the tertiary structure of proteins and, therefore, can be
used to dissociate antigen-antibody complexes. Chaotropic salts disrupt ionic
interactions, hydrogen bonding, and sometimes hydrophobic interactions.
Chaotropic anions are effective in the order SCN->ClO
4
->I->Br->Cl-. Chaotropic
cations are effective in the order guanidine>Mg 2 +>K+>Na+. Eluants such as urea
(up to 8 M), guanidine-HCl (up to 6 M), and NaSCN (up to 6 M) are effective in
disrupting most protein-protein interactions. However, these strong chaotropic
agents may destroy the activity of the antigen, the antibody, or both. Conditions as
mild as possible should always be used.
It is important to remove the eluted antigen or antibody from eluant as quickly as
possible to minimize the chance of denaturation. If acid or base is used, the
samples should be neutralized immediately following elution. If a chaotropic agent
is used for elution, it can be rapidly removed by desalting (Econo-Pac desalting
columns, Econo-Pac P6 desalting cartridges, Bio-Gel
® P-6 desalting gel, or for
very small volumes, Micro Bio-Spin columns).
Section 5
Renaturation of Eluted Proteins
Proteins that have been denatured during elution can often be renatured by the
addition of a chaotropic agent such as guanidine-HCl, followed by stepwise
dialysis against decreasing concentrations of the chaotrope. The high
concentration of guanidine-HCl puts the protein into a random coil configuration.
As the chaotrope is slowly removed, the protein will return to its native form.
Section 6
Assessing Protein Purity
Check the integrity of purified protein by SDS-PAGE analysis (such as on
Criterion
™ or Criterion ™ XT precast midi gels). Samples containing guanidine-HCl
cannot be run directly on these gels due to precipitation of protein. First remove
the guanidine by desalting using Bio-Gel P-6 (or -30) spin columns.
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