Bio-Rad Affi-Gel Hz Hydrazide Gel - Manual (Page 12)

5.2 Sample Application
1. Sample is applied to the immobilized IgG column. Samples
should be free of particulates. Complex samples should be dilut-
ed in application buffer and filtered if necessary. This will
enhance specific binding to the immobilized IgG and prolong
column life.
2. Wash column with 2 bed volumes of 0.5 M NaCl in application
buffer to remove any unbound protein.
3. Wash column with 1-2 bed volumes of application buffer of
lower NaCl concentration. The column is now ready for elu-
tion of bound antigen.
5.3 Elution Suggestions
The elution conditions necessary to break the antibody-antigen
bond vary according to bond strength. Elution conditions listed are
suggestions, and optimal conditions should be determined empiri-
cally. When choosing elution schemes for affinity purification, select
conditions which give satisfactory purification without damaging the
matrix or the product. Very harsh conditions may denature the anti-
body coupled to Affi-Gel Hz gel and affect column performance.
Start with conservative rather than severe conditions and optimize
elution with slight modifications from run to run. In general, elution
should be carried out quickly. Request bulletin 1099 for further dis-
cussion of elution schemes.
1. Add 2 bed volumes of eluant to affinity column.
2. Collect fractions and/or monitor elution profile with UV-visible
detector. The eluted antigen should be neutralized if eluted in low
pH, or precipitation may occur.
3. Allow the elution buffer to reach the top of the gel bed. Quickly
regenerate the column in application buffer containing 0.02%
sodium azide, and store at 4 °C until next use.
4. Quantitate purification yield.
Acid Elution
0.2 M glycine-HCl, pH 2.5
0.1 M acetic acid
0.15 M sodium citrate, pH 3.0
0.5 M formic acid
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