Bio-Rad Affi-Gel 15 Gel - Manual (Page 16)

Optimizing Support and Sample Volumes
Use only the required amount of affinity support. If
excess support is used, sample elution becomes more dif-
ficult because the sample continues to bind and elute as it
passes down the column. Stronger elution conditions
become necessary, residence time is longer, the eluted
peak is broader, and there is a greater risk of denaturation
and poor recovery.
One method to insure that only the required amount of
affinity gel is used is to apply the sample to the top of the
column and elute using reverse flow (see Figure 5).
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Section 9
Immunoaffinity Chromatography
with Affinity Supports
9.1 Adsorption of the Sample
Preparing Antiserum for Antibody Purification
If an antibody from serum is to be affinity purified on
an immobilized antigen support, partial purification of the
antiserum is recommended. DEAE Affi-Gel blue gel and
CM Affi-Gel blue gel will remove complement factors
which bind immune complexes. They will also remove
protease which can destroy valuable antibody during sam-
ple storage or decrease column life by destroying the
immobilized protein. Antiserum should at least be heat
inactivated at 56 °C for 30 minutes to destroy the comple-
ment factors.
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