Bio-Rad Affi-Gel Hz Hydrazide Gel - Manual (Page 11)

= (mg IgG/ml) x dilution factor x sample volume = total IgG
[total protein before coupling] - [total uncoupled protein (eluant + 0.5 M NaCl
wash)]
= [total coupled protein]
x 100 = % protein coupled
The starting and final IgG solutions and 0.5 M NaCl wash can
be analyzed with the Bio-Rad Protein Assay to determine total pro-
tein as an alternative to the absorbance method or for IgG samples
of less purity.
Section 5
Applications of Affi-Gel Hz
Immobilized IgG
In this section, suggestions for sample application and elution
are presented. Conditions listed are commonly employed for elution
in affinity purification. These are by no means the only elution con-
ditions which may be used. When choosing elution conditions for
your application, refer to Section 5.3 and Section 6 on precautions
and product specifications. Elution conditions should facilitate sat-
isfactory purification without damaging the affinity column or prod-
uct.
5.1 Conditioning the Immunoaffinity Column
It is necessary to condition the column prior to applying the
sample mixture.
1. Remove column from 4 °C and allow it to reach room temper-
ature. Add 2-4 bed volumes of the buffer chosen for antigen
elution to the affinity column.
2. Regenerate the column with at least 5 bed volumes of applica-
tion buffer (such as PBS, pH 7.0). The immunoaffinity column
is now ready for sample application.
9
(total coupled protein)
Abs. @ 280 nm
1.4
(total protein before coupling)
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