Bio-Rad Affi-Gel Protein A Media - Manual (Page 6)

471 g for 1,500 ml.) Stir for 10 minutes. Filter through a 0.22 µ
nylon filter and check the pH. The pH should be 9.0 ± 0.2. If the pH
is not in this range, adjust the pH with 10 N NaOH or 6 N HCl.
Store buffer solids at room temperature. Store reconstituted buffer at
4 °C. If desired, sodium azide may be added to 0.05% (w/v).
Elution Buffer Preparation
The elution buffer is supplied as a preweighed, premixed solid.
Reconstitution and filtration are required prior to use. These salts
are hygroscopic. Any material in clumps should be broken up
before you weigh any solids. Dissolve 2.2 g per 100 ml distilled
deionized water (use the full 25 g for 1,100 ml). Stir for 10 minutes.
Filter through a 0.22 µ filter and check the pH. The pH should be
3.0 ± 0.2. If the pH is not in this range, adjust the pH with 10 N
NaOH or 6 N HCl. Store buffer solids at room temperature.
Reconstituted buffer should be stored at 4 °C, and if desired,
Thimerosal bacteriostat may be added to a final concentration of one
part per ten thousand. The use of sodium azide in low pH buffers
is not recommended.
Sample Preparation
Ascites samples should be diluted one to one with binding
buffer. If your sample is tissue culture supernatant, it should be con-
centrated to approximately 5 mg immunoglobulin/ml and then dilut-
ed one to one with binding buffer.
Standard Mouse IgG
1
Purification
Procedure
1. Pack a 1 x 10 cm Econo-Column chromatography column (sup-
plied) with the desired volume of Affi-Gel protein A agarose.
(One ml of Affi-Gel protein A agarose will bind 6-8 mg/ml
mouse IgG
1
.)
2. Equilibrate the column with 5 bed volumes of binding buffer.
After equilibration, the pH of the column effluent shold be equal
to the pH of the binding buffer (pH 9.0).
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