Bio-Rad Affi-Gel 15 Gel - Manual (Page 20)

Alternative procedures have been published for elut-
ing labile antigens from immobilized antibody columns.
Deionized water has been reported,
8,9 but yields are gener-
ally low. A method of increasing interest is electrophoretic
elution.
1 An electrical field is applied and the adsorbed
protein is electrophoresed away from the affinity matrix.
9.5 Renaturation of Eluted Proteins
Proteins which 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.
If you have any questions or suggestions regarding
the use of this or any of our other products, contact your
local Bio-Rad representative. Inside the United States, call
1-(800)-4BIORAD.
35
samples should be neutralized immediately following elu-
tion. If a chaotrope is used for elution, it can be rapidly
removed by desalting (Econo-Pac
® 10DG desalting
columns, Bio-Gel ® P-6 DG desalting gel, Econo-Pac P6
desalting cartridges, or for very small volumes Bio-Spin ®
columns).
9.4 Special Considerations for Labile
Antigens
The stability of some antigens can be a problem.
Special considerations may be necessary. The mildest elu-
tion conditions possible are desirable, with rapid elution
and short exposure times being critical. For labile
immobi-
lized antigen relatively mild elution conditions can be
used, and then a more complete regeneration with a
chaotropic salt can be done after every fourth or fifth use
of the column. This will increase the life of the column by
minimizing exposure to stringent conditions, and strip the
column of bound proteins to maintain the capacity.
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