Manual

Optional

Bio-Rad Protein Assay Kit I (catalog number 500-0001)

Section 2

Monoclonal Antibodies

Due to the unique specificity of monoclonal antibodies, cou-

pling to Affi-Gel Hz gel may not be optimal for all monoclonal

antibodies. Loss of activity may occur depending on the individual

monoclonal. To minimize loss of valuable antibody preparations, Bio-

Rad highly recommends that a sample coupling experiment be per-

formed to determine the efficiency of the coupling reaction. After

the results of the sample coupling are determined, the remaining

antibody can be coupled to the Affi-Gel Hz gel. If results are less than

adequate with the Affi-Gel Hz gel, use Affi-Gel 10 gel to immobi-

lize the monoclonal antibody.

Section 3

Antibody Purification

Prior to IgG coupling to Affi-Gel Hz hydrazide gel, the anti-

body must be at least partially purified. A high degree of purity will

insure that higher IgG concentrations are available for oxidation

and immobilization to the affinity support. This increased purity of

IgG will maximize coupling capacity and purification potential.

There are many approaches to antibody purification, and only a few

will be presented here.

For use with this kit, the purified antibody should be in a total

protein concentration of 1-5 mg/ml in a total maximum volume of

5 ml prior to buffer exchange. This recommended volume is nec-

essary for rapid desalting and buffer exchange with the Econo-Pac

10DG columns. Regardless of the purification method(s) chosen, it

will be necessary to buffer exchange the purified antibody into the

Affi-Gel Hz coupling buffer prior to oxidation and coupling fol-

lowing the procedure in this manual. See Buffer Exchange, Section 4.1.

IgG from serum or ascites can be purified by using a combina-

tion of ion exchange with the Macro-Prep

high S support and

hydrophobic interaction chromatography with the Macro-Prep

t-butyl support (or a combination of affinity chromatography

with CM Affi-Gel blue gel and hydroxyapatite chromatography

with Macro-Prep CHT, type I support). DEAE Affi-Gel blue chro-

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