Bio-Rad Bio-Scale™ Mini Affi-Prep® Protein A Cartridges - Manual (Page 6)

Note: For many murine IgGs the capacity is as high as 10 mg antibody/ml Affi-Prep protein
A or 5 mg antibody/Affi-Prep protein A analytical cartridge.
5. Continue flow at 0.5 ml/minute until the unbound fraction of contaminating proteins is
completely removed from the matrix (typically within 10 column volumes) and the UV-
absorbance trace returns to baseline.
6. Change to Affi-Prep MAPS II elution buffer and return flow to 0.5 ml/minute. The IgG will
typically elute at, or slightly behind, the elution buffer/binding buffer interface.
Note: For many samples, a less acidic release buffer is sufficient. If a particular IgG is acid
sensitive, alternative elution buffers should be tested.
7. Store the Affi-Prep cartridge in a mild, neutral storage buffer. Recommended is 0.1 M
HEPES, 1 mM EDTA, pH 7.5, containing 0.02–0.05% sodium azide.
8. Flush buffer pump(s) with storage buffer or water prior to system shutdown. This greatly
reduces the possibility of damage to non-resistant fluid paths, since MAPS II buffers contain
halide salts that can form deposits on standing.
Section 3
Answers to Common Questions
1. Regeneration of the cartridge:
Affi-Prep protein A matrix need not be regenerated. Affi-Prep protein A matrix has been
recycled reproducibly up to 550 times without regeneration.
If regeneration is desired, regenerate the Affi-Prep protein A cartridge with 50% methanol
after every use. The cartridge can be washed with 0.1 N NaOH every 5-10 runs for a more
stringent wash. This NaOH wash should only be used after the regular methanol
regeneration step. For complete sanitation (i.e., removal of endotoxins and DNA) the
cartridge can be washed with 1.0 N NaOH.
If the cartridge will be re-used right away, re-equilibrate the cartridge with at least 5
cartridge volumes of MAPS binding buffer. If the cartridge is to be stored, equilibrate the
cartridge with a mild neutral buffer such as 50 mM sodium phosphate, pH 7.5, containing
0.02–0.05% sodium azide.
2. Sensitivity of antibodies to low pH:
Some antibodies can be inactivated by low pH. Inactivation may be reduced by collecting
the eluted immunoglobulin fraction into a concentrated, high pH buffer, or the fraction can
be dialyzed against a buffer of choice. In cases of extreme sensitivity, many
immunoglobulins can be eluted at pH 4-6 by raising the pH of the elution buffer with 1.0 N
NaOH.
3. Flow rate of the Affi-Prep protein A analytical cartridge:
Maximum flow rate is 2.0 ml/minute; 0.5 ml/minute is recommended for maximum binding
efficiency for viscous samples (e.g., ascites fluid).
4. Purification of IgG from species other than mouse:
When the specially optimized Affi-Prep protein A MAPS II buffers are used in conjunction
with the Affi-Prep protein A matrix, a broad range of monoclonal and polyclonal antibodies
can be purified. Representative capacities for porcine, canine, bovine, murine, and rabbit are
in the range of 9-16 mg polyclonal IgG per ml matrix.
5. Shelf life of the Affi-Prep protein A MAPS II kit:
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