Bio-Rad Affi-Gel Hz Hydrazide Gel - Manual (Page 6)

matography may also be used to remove most serum components
except transferrin.
IgG in ascites fluid and serum may be purified by Affi-Gel pro-
tein A agarose (MAPS ® II kit) low pressure chromatography, or by
application to Affi-Prep ® protein A medium to high pressure poly-
meric matrix. Protein A, a surface protein from Staphylococcus
aureus, binds the Fc region of many mammalian IgG species. Protein
A purification will yield highly purified IgG for coupling to Affi-Gel
Hz gel.
Hybridoma tissue culture supernatant containing dilute anti-
body (µg/ml concentrations) will necessitate a scheme to concentrate
and purify the immunoglobulin. Ammonium sulfate precipitation fol-
lowed by dialysis and ion exchange chromatography, Bio-Gel
® HT
hydroxyapatite, or Affi-Gel protein A gel are options that will yield
highly purified, concentrated antibody.
Some immunoglobulins are sensitive to high ammonium sul-
fate concentrations or low pH and, as a result, total antibody activ-
ity can be irreversibly reduced prior to coupling to Affi-Gel Hz gel.
Care must be taken to minimize the duration of antibody exposure
to these conditions to retain activity essential to the applications of
the immunoaffinity matrix.
Section 4
Immobilization Protocol
4.1 Buffer Exchange
Prior to coupling to Affi-Gel Hz gel, it is necessary to exchange
the buffer in which the purified antibody appears. The Affi-Gel Hz
coupling buffer is optimized for antibody oxidation and immobi-
lization to Affi-Gel Hz hydrazide gel. This buffer exchange can be
easily performed using the Econo-Pac 10DG desalting columns
provided.
Antibody immobilization to Affi-Gel Hz gel consists of buffer
exchange prior to the oxidation of the carbohydrate moieties on the
Fc region, desalting, and coupling. This oxidation forms aldehydes
for binding to the hydrazide functional group on the gel. Since the
carbohydrate constitutes a small percentage of the total glycoprotein
weight, the coupling reaction will progress slower than activated, self-
coupling gels. The bond formed is a stable hydrazone linkage that
is chemically resistant to many common elution conditions employed
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