CM Affi-Gel®Blue Gel
Instruction Manual
Catalog Number
153-7304
Introduction
o
NH
2
o
NH
SO2ONa
NH
N
N
N
O
Crosslinked
Agarose
NH
R
1
R
2
R1 = H or SO2ONa
R
2
= SO2ONa or H
CM Affi-Gel blue gel is a bifunctional affinity/ion exchange
chromatography matrix prepared by coupling Cibacron blue
F3GA and carboxymethyl (CM) groups to Bio-Gel®A-5m
crosslinked agarose gel. The Cibacron blue functions as an ionic,
hydrophobic, or sterically active binding site for proteins with
dinucleotide folds, such as albumin.
Cibacron blue coupled to agarose.
The carboxymethyl functional group functions as a cation
exchanger and will bind proteins with isoelectric points lower
than the pH of the mobile phase. (At pHs below 4.5 the carboxymethyl group will be protonated and will not function as an
ion exchanger.) This bifunctionality allows CM Affi-Gel blue
gel to bind both albumin and protease from serum. Dialysis of
1
serum is not necessary prior to chromatography on this gel.
Albumin and serum protease are bound strongly enough that a
high concentration of salt or chaotropic reagent is required to
elute these components. Chromatography on CM Affi-Gel blue
gel provides a convenient initial step in the purification of serum
proteins.
Product Description
Matrix Bio Gel A-5m agarose gel
Particle size 150–300 µm (50-100 mesh)
Shipping medium 50 mM KCl with 0.04% NaN
Functional groups Cibacron blue and carboxymethyl
Flow rate 15–25 cm/hr
Pressure limit 15 psi
Capacity
Serum 2 to 6 ml of gel/ml of serum (lot
Stability
pH 2–11
Organic solvents alcohols
Temperature autoclavable
Storage 1 year at 4 °C, in 0.02% NaN
dependent)
other preservative
3
Material required but not supplied
Pre-wash buffer 0.1 M acetic acid, pH 3, 1.4 M NaCl,
Running buffer 10 mM K
Regeneration buffer 2 M guanidine HCl or 1.5 M NaSCN
Buchner funnel
Chromatography column
40% isopropanol
NaCl, 0.02% NaN
HPO4, pH 7.25, 0.15 M
2
,
3
General Instructions
1. Prepare CM Affi-Gel blue gel by washing it on a Buchner
funnel, or in a column, with 5 bed volumes of pre-wash
buffer, followed by 7 bed volumes of deionized water. This
is required the first time the gel is used, or if the gel has been
stored for more than 1 week since the previous use. Small
amounts of the blue dye may appear in the alcohol wash.
After prewash, equilibrate the column with 3 bed volumes
of running buffer.
The capacity of the gel is lot dependent, and will range
between 2–6 ml of gel per ml of serum. The capacity for
or
3
human and rabbit serum for a specific lot of gel is printed on
the label on the bottle.
**
32
*
2. To insure that the prewash removed excess dye in the gel,
wash the gel with 2 bed volumes of 1.4 M NaCl and then
with running buffer. If the high salt wash is colored, repeat
the first pre-wash.
3. Pack the gel into a suitable column.
4. Apply the serum sample.
5. Wash the column with 2 bed volumes of running buffer at a
flow rate of 15–30 cm/hr. The effluent from this step contains the serum proteins minus plasminogen and albumin.
Approximately 90% of the globulin will elute in one column bed volume.
6. The albumin can be eluted with 2 bed volumes 1.4 M NaCl
in running buffer. This is optional, and can be eliminated.
7. Whether the albumin is eluted or not, regenerate the column
with 2 bed volumes of regeneration buffer followed by 2
bed volumes of running buffer.
Note: The first one or two cycles of the gel may show low levels
of eluted dye in the high salt peak. This does not affect the functionality of the gel.
8. 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. The useful life of
the gel is generally eight to ten cycles.
Protease Free Globulin Fraction from
Serum with CM Affi-Gel Blue Gel
A partially purified globulin fraction is often desirable in the
preparation of immunological reagents. This is commonly
achieved by a two-step procedure involving fractionation on
DEAE anion exchange chromatography followed by ammonium
sulfate precipitation.1Recovery of active antibody by this method
is generally about 65%, and detectable protease activity remains.
In fact, ammonium sulfate may serve to activate serum proteases.
Better recoveries (80–90%) and complete removal of protease are
achieved by using CM Affi-Gel blue affinity gel instead of DEAE
cellulose, followed by an ammonium sulfate precipitation as an
additional purification and concentration step to obtain a globulin
fraction free of protease and serum complement proteins. This
procedure is particularly useful when processing large volumes of
serum or when high yields (>90%) are desired.
2
4
5
Instructions for a Protease Free
Globulin Fraction From Serum
1. Follow the general instructions through step 5.
2. Measure the total volume of the collected protein peak.
Determine the amount of solid ammonium sulfate required
to achieve a 45% saturated solution.
pooled protein with a stir bar on a stir plate, slowly add the
ammonium sulfate to the solution.
3. After all the (NH4)2SO4has been added, continue stirring
for at least 1 hour at room temperature. If stirred or stored
overnight before centrifugation, the suspension should be
kept at 4 °C.
4. Centrifuge the suspension in a refrigerated (4 °C) centrifuge
at approximately 1,000 g for 20 minutes. (If supernatant does
not appear clear, centrifuge longer.) Discard supernatant.
5. Resuspend the pellet in 45% saturated ammonium sulfate
by breaking up pellet with a pipet and centrifuge again.
6. Resuspend pellets in a minimum volume of running buffer.
Do not mix vigorously to dissolve pellets (this will denature
proteins). If pellet does not go into solution readily, let sit in
PBS at 4 °C for 1–2 hours, and then mix.
***
While stirring the
7. Remove any remaining ammonium sulfate, by desalting on
Bio-Gel P-6DG desalting gel or prepacked desalting
columns using gravity flow, with the Econo-Pac®P6 cartridge, or by dialysis.
8. Continue with step 6 of the general instructions for CM
Affi-Gel blue gel column regeneration.
6
7
References
1. Hudson, L. and Hay, F. C., Practical Immunology, p. 152,
Blackwell Scientific Publications, Oxford, England (1976).
2. Steinbuch, M., Audran, R. and Pejaudier, L., C. R. Soc. Biol., 164,
296 (1970).
3. Gee, A. P., Borsos, T. and Boyle, M. D. P., J. Immunol. Methods,
30, 119 (1979).
4. Kelleher, P C., Smith, C. J. and Parnell, R., J. Chromatog., 173,
415 (1979).
5. Gianazza, E. and Arnaud, P., Biochem. J., 201, 129 (1982).
6. Ledden, D. J., Feldhoff, R. C. and Chan, S. K., Biochem. J., 205,
331 (1982).
Cibacron is a registered trademark of Ciba-Geigy.
*** Note: If this alcohol wash is done in a column you may notice
shrinkage of the gel.
*** For species other than human or rabbit, use the higher gel/serum
level indicated on the CM Affi-Gel blue gel label.
*** 275 grams (NH
4)2SO4
per liter.
Ordering Information
Catalog
Number Product Description
153-7304 CM Affi-Gel Blue Gel, 100 ml
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 10DG Desalting Columns, 10 ml, 30
732-0011 Econo-Pac P6 Cartridge, 5 ml
8
9
Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547
LIT308 Rev C
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