DEAE Affi-Gel®Blue Gel
Instruction Manual
Catalog Number
153-7307
Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
4006049 Rev A
Introduction
DEAE Affi-Gel Blue gel is a bifunctional affinity/ion
exchange chromatography matrix prepared by coupling
Cibacron
Bio-Gel
functions as an ionic, hydrophobic, or sterically active
binding site for proteins with dinucleotide folds, such as
albumin. The diethylaminoethyl functional group
functions as an anion exchanger and will bind proteins
with isoelectric points higher than the pH of the mobile
phase. This bifunctionality makes DEAE Affi-Gel Blue
gel a very powerful tool. It is possible to obtain highly
purified IgG from serum from a wide variety of species
through carefully optimizing the ionic strength and pH of
the application buffer.
provides a convenient initial step in the purification of
serum proteins. After the IgG has eluted, additional
fractions can be obtained from the DEAE Affi-Gel Blue
gel by elution with an ionic strength gradient. DEAE
Affi-Gel Blue gel has a greater binding affinity for serum
albumin than DEAE ion exchangers, and offers a
®
blue F3GA and diethylaminoethyl groups to
®
A-5m, agarose gel. The Cibacron blue F3GA
Chromatography on DEAE Affi-Gel Blue gel
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superior method of obtaining serum fractions
uncontaminated by albumin.
Product Description
Matrix Bio Gel A-5m agarose gel
Particle size 150-300 µm (50-100 mesh)
Shipping medium 0.01M Tris, pH 8, 0.15 M NaCl,
Functional Groups Cibacron blue and diethylaminoethyl
Typical flow rate* 15-25 cm/hr
Pressure limit 15 psi
Capacity**
Serum 0.2-1 ml of serum/ml of gel
Recovery of IgG >55%
Recovery of Albumin >90%
Removal of protease 100%
Stability
pH 2-11
Organic solvents alcohols
Temperature not autoclavable
Storage 1 year at 4 °C, in 0.02% NaN
*Flow rate determined using a 1.5 x 20 cm column, and a hydrostatic
pressure of 1:1.
**The capacity for rabbit serum and human serum is lot to lot dependent.
It is determined on every lot, and provided on the label.
0.04% NaN
other preservative
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Material Required but not Supplied
Pre-wash buffer 0.1 M acetic acid, pH 3, 1.4 M NaCl,
Running buffer see Table 2
Regeneration buffer 2 M guanidine HCl in application
Buchner funnel
Chromatography column
40% isopropanol
buffer, or 1.5 M NaSCN
General Instructions
1. Prepare the appropriate buffer using the information
given in Table 1. Accurate buffer preparation is
essential for optimum IgG recovery. See Optimizing
the Separation Conditions.
Table 1. Buffers for Purification of IgG from Serum on DEAE AffiGel Blue gel
Species Application Buffer
Rabbit 20 mM Tris-HCl, pH 8.0, 25 mM NaCl,
or
Sheep as for rabbit
Goat as for rabbit
Human* 20 mM K
Mouse 20 mM Tris-HCl, pH 7.2, 25-50 mM NaCl
These buffers are recommended starting points. Minor changes may be
needed. See Optimizing the Separation Conditions.
is used to prepare this buffer. The pH is adjusted with KOH.
*KH
2PO4
0.02% NaN
3
HPO4, pH 8.0, 0.02% NaN
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3
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2. Transfer the serum sample to the buffer. This should
be done using Bio-Gel P-6DG for buffer exchange,
or through dialysis.
3. Rinse the gel on a Buchner funnel with a least 5 bed
volumes of pre-wash buffer. DEAE Affi-Gel Blue
gel has an excess of dye. This wash will elute
residual dye which might be eluted in serum protein
fractions. Continue rinsing with at least 10 bed
volumes of application buffer (Table 1) to insure that
the ionic strength is lowered. Note: If the alcohol
wash is done in a column, you may notice shrinkage
of the gel.
4. Prepare a column of DEAE Affi-Gel Blue gel using
the gel-to-serum volume ratio given on the bottle
label. Calculate the total volume of gel needed using
the initial serum volume, prior to buffer exchange or
dialysis.
The capacity of the gel is lot dependent, and will
range between 0.2 and 1 ml of serum per ml of gel.
The capacity for human and rabbit serum for a
specific lot of gel is printed on the label of the gel
bottle. For species other than human or rabbit, use
the lower capacity given.
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5. Elute the column with three volumes of application
buffer.
6. Apply the serum sample and elute with three bed
volumes of starting buffer. Collect fractions which
are approximately the volume of the applied sample.
7. Beginning with the first tube of unbound protein
peak, combine effluent tubes to give a total volume
equivalent to eight times the volume of the initial
serum sample.
8. For the isolation of other serum proteins, the column
may be eluted with a gradient of increasing salt
concentration. A final NaCl concentration of 0.5 M
is usually enough to elute all serum proteins except
albumin.
9. Most of the bound albumin can be eluted by washing
the column with 2 to 3 bed volumes of 1.4 NaCl in
application buffer, or with regeneration buffer.
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10. Regardless of whether the albumin is eluted or not,
regenerate the column with 4 to 5 bed volumes of
regeneration buffer followed by 3 bed volumes of
application buffer.
11. Some loss of capacity due to small amounts of
protein which remain bound to the gel may be
evident after about five cycles. To compensate for
this, increase the gel-to-sample ratio by 20% for
subsequent cycles. For serum preparations, the
useful life of the gel is generally eight to ten cycles.
Note: The first one or two runs using the gel may show
low levels of eluted dye in the high salt peak. This dye
does not interfere with the usefulness of the gel and
should not appear in subsequent runs. Leaching of the
dye can be avoided by washing the column with the
prewash buffer.
Optimizing the Separation
Conditions
The end result is highly dependent on the pH and
ionic strength of the sample. It is crucial that serum is
transferred into the appropriate application buffer, either
through desalting using the Bio-Gel P-6DG gel, or
through dialysis. The buffers listed in Table 1 have
provided complete plasminogen removal when tested,
but some variation in non-IgG protein binding and IgG
recovery may be encountered using different serum
preparations. For this reason, the unbound protein
fraction, ideally containing only IgG, should always be
tested for proteolytic activity as described above. If a
sample displays minor amounts of proteolytic activity in
the unbound protein fraction, it may be necessary to
modify the buffer. This experiment may also help to
determine conditions for even higher recoveries of IgG in
the unbound fraction.
Transfer a sample to 20 mM Tris-HCl, pH 8.0, and
apply it to a column equilibrated with the same buffer.
Elute the column with a sodium chloride gradient, and
collect fractions of the same volume as the sample
applied. The sodium chloride concentration at which
plasminogen elutes can be determined by testing the
effluent for proteases. Fractions free of protease can be
pooled, or a second serum sample can then be purified at
a sodium chloride concentration slightly lower than that
at which non-IgG protein is detected. For higher purity or
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improved recovery of IgG, use Affi-Gel Protein A
support.
For more information and applications, request
Bulletin 1092.
Catalog
Number Product Description
153-7307 DEAE Affi-Gel Blue Gel
For desalting and sample preparation
150-0738 Bio-Gel P-6DG Desalting Gel, 100 g
150-0739 Bio-Gel P-6DG Desalting Gel, 1 kg
732-2010 Econo-Pac
30 x 10 ml
732-0011 Econo-Pac P-6 Cartridge, 5 ml
Other products for IgG purification
732-0031 Econo-Pac DEAE Cartridge, 1 x 5 ml
732-0035 Econo-Pac DEAE Cartridge, 5 x 5 ml
732-2026 Econo-Pac Serum IgG Purification Columns
732-2027 Econo-Pac Serum IgG Purification Kit
Cibacron is a registered trademark of Ciba-Geigy.
®
10DG Desalting Columns,
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