Bio-Rad Bio-Scale™ Mini Affi-Prep® Protein A Cartridges - Manual (Page 5)

Section 2
Instructions for Use
2.1 Buffer Preparation
Binding Buffer Preparation
The binding buffer is supplied as a premixed, preweighed solid. Dissolve 31.4 gm binding
buffer solids to a final volume of 100 ml using distilled, deionized water. Use the full 471 gm for
1,500 ml. Stir for 10 minutes, or until fully dissolved. Filter through a 0.22 µm membrane filter
and check the pH. The pH should be 9.0 ± 0.2. Store 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 hydroscopic. Any material in clumps should be broken
up before weighing the solids. Dissolve 2.2 g to a final volume of 100 ml using distilled,
deionized water. Use the full 25 g for 1,100 ml. Stir for 10 minutes, or until fully dissolved.
Filter through a 0.22 µm filter and check the pH. The pH should be 3.0 ± 0.2. Buffer should be
stored at 4 °C.
2.2 Sample Preparation
Proper adjustment of the pH and ionic strength of the sample is critical for optimal binding.
For best results, the sample pH should be adjusted to 9.0, and the ionic strength of the sample
should approach that of the MAPS binding buffer. This can be achieved by sample dilution,
dialysis, or buffer exchange using the Econo-Pac
® 10DG desalting columns, Econo-Pac P6
cartridges, or Bio-Gel ® P-6DG gel filtration gel.
• Ascites fluid should be diluted 1:2 with binding buffer. Higher concentrations of binding
buffer can enhance the binding of low affinity antibodies.
• Tissue culture supernatant may be concentrated to approximately 5 mg immunoglobulin
per ml, and then diluted 1:2 with binding buffer. For large volume samples where further
dilution is not desired, we recommend adding the dry binding buffer salts directly to the
sample instead of diluting the sample with prepared buffer.
• All samples should be filtered through a 0.45 or 0.8 µm filter before loading onto the
cartridge.
2.3 Standard Mouse IgG Purification Procedure
1. Place the Affi-Prep protein A analytical cartridge into the Standard Cartridge Holder,
making sure that the writing on both are aligned in the same direction. Tighten the end nuts
of the holder.
2. Place this assembly in-line in the HPLC or medium pressure system.
Note: Direction of buffer flow is in the same direction as the lettering on both the analytical
cartridge and holder. The inlet side is on the left as you hold the cartridge assembly and read
the Bio-Rad logo.
3. Equilibrate the Affi-Prep protein A analytical cartridge with Affi-Prep MAPS II binding
buffer for 15 minutes at 0.5 ml/minute (15 column volumes).
4. Dilute the IgG containing sample 1:1 or 1:2 with binding buffer (i.e., 1 volume sample plus
1 or 2 volumes buffer). Filter through a 0.45 µm or 0.8 µm membrane just prior to sample
loading. Inject a sample containing up to 2.5 mg of IgG onto the cartridge.
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LIT79B 9/1/98 8:12 AM Page 3