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Affi-Prep®Protein A Matrix
Instruction Manual
Catalog Numbers
156-0005
156-0006
Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
LIT-230 Rev B
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Table of Contents
Section 1 Introduction ..........................................1
Section 2 Product Information.............................4
Section 3 Ordering Information ..........................5
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Section 1
Introduction
Affi-Prep protein A gel consists of highly purified
protein A covalently coupled to a unique macroporous
polymer matrix. This support is intended for use in
medium to high pressure chromatographic applications.
All specifications have been developed using the AffiPrep protein A MAPS
6164).
The usefulness of protein A purification for murine
monoclonal antibodies has been limited because, with
published methods, most IgG
significant purification problem. The MAPS buffer
system for protein A affinity chromatography was
developed to optimize the binding and recovery of many
immunoglobulins, especially mouse monoclonal
antibodies. The MAPS buffer system has been shown to
increase protein A’s capacity for IgGs and IgMs from
many different species. Approximately 50% of all IgMs
will bind to protein A when the MAPS buffer system is
used. This buffer system is recommended for use with all
protein A affinity supports.
®
II buffers (catalog number 156-
retention represents a
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Sample Preparation
Proper adjustment of the pH and ionic streng h 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
desalting columns or Bio-Gel
®
P-6DG gel filtration gel.
®
10DG
• Ascites fluid should be diluted 1:2 with binding
buffer. Higher concentrations of binding buffer can
enchance the binding of low affinity antibodies.
• Tissue culture supernatant should 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 column.
Purification Protocol
1. Pack a suitable chromatography column with the
desired volume of the Affi-Prep protein A support.
2. Equilibrate the column with 5-10 bed volumes of
Affi-Prep MAPS II binding buffer. After
equilibration, the pH of the column effluent should
be equal to the pH of the binding buffer (pH 9.0).
3. Apply the prepared sample to the column.
4. Wash the column with 10-15 bed volumes of binding
buffer to remove all of the unbound contaminating
components.
5. Elute the immunoglobulin with 5 bed volumes of
Affi-Prep MAPS II elution buffer. Elute with and
additional 10 bed volumes of elution buffer to insure
total removal of immunoglobulin. Neutralize the
eluted sample immediately after elution with 1 M
Tris-HC1. (Prolonged exposure of the purified
immunoglobulin fraction to acid pH should be
avoided.)
6. Regenerate the Affi-Prep protein A column with
50% methanol after every use. The column can be
washed with 0.1 N NaOH every 5-10 runs for a more
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stringent column 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 support can be washed
with 1.0 N NaOH. This is an acceptable method of
sanitation for FDA purposes.
7. If the column will be re-used right away, re-
equilibrate the column with at least 5 column
volumes of MAPS binding buffer. If the column is to
be stored, equilibrate the column with a mild neutral
buffer such as 0.05 M sodium phosphate, pH 7.5,
containing 0.02-0.05% sodium azide.
Section 2
Production Information
Monoclonal antibody Mouse IgG1= 8-10 mg/ml
loading capacities IgG
Polyclonal antibody Human IgG = 16-23 mg/ml
= 13-15 mg/ml
2a
IgG
= 13-15 mg/ml
2b
IgG
= 8-10 mg/ml
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loading capacities Rat, sheep, bovine, equine,
goat, rabbit, dog, and porcine
IgG = 9-16 mg/ml
IgM = 5-7 mg/ml*
Flow rate 2,000 cm/hr maximum; 150
cm/hr recommended. We
recommend the use of the
Bio-Rex
®
MP
chromatography columns for
medium pressure Affi-Prep
protein A applications.
Pressure limit 1,000 psi
Chemical stability pH 2-14, 1 N NaOH, 8 M
urea, 50% methanol, 6 M
guanidine-HC1
Shipping buffer 0.05 M sodium phosphate,
pH 7.5, containing 0.05%
sodium azide
Shelf life 1 year at 4 °C
*Approximately 50% of all IgM’s will bind to protein A.
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Section 3
Ordering Information
Catalog
Number Product Description
156-0006 Affi-Prep Protein A Support, 5 ml
156-0005 Affi-Prep Protein A Support, 25 ml
153-6164 Affi-Prep Protein A MAPS Buffers, (contains 1.5 L
binding and 1.1 L elution buffers)
153-6161 Affi-Prep Protein A MAPS II Binding Buffer, 5
liters (contains enough solids to make 5 liters of
binding buffer)
153-6162 Affi-Prep Protein A MAPS II Elution Buffer, 5
liters (contains enough solids to make 5 liters of
elution buffer)
Affi-Prep Protein A Prepacked HPLC Cartridges
and Columns
153-6165 Affi-Prep Protein A MAPS Kit, contains 1 MAPS
II buffers (153-6164), 1 Affi-Prep protein A MAPS
analytical cartridge (125-0460), and 1 Standard
Cartridge Holder (125-0131)
125-0460 Affi-Prep Protein A MAPS Analytical Cartridge,
30 x 4.6 mm
125-0131 Standard Cartridge Holder, for 30 x 4.6 mm
cartridges
Catalog
Number Product Description
125-0461 Affi-Prep Protein A MAPS Preparative
Cartridge, 15 x 25 mm
155-0130 MAPS Preparative Cartridge Holder, for 15 x 25
mm cartridges
155-0001 Affi-Prep Protein A MAPS Preparative Column,
100 x 25 mm
155-0005 Affi-Prep Guard Cartridge, 15 x 25 mm, for all 25
mm ID columns and cartridges
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