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Affi-Gel®Blue Gel
Instruction Manual
Catalog Numbers
153-7301
153-7302
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Table of Contents
Section 1 Introduction............................................ 1
Section 2 Product Description............................... 3
Section 3 Instructions for Use ............................... 4
3.1 Sample Preparation.......................................... 4
3.2 Albumin Removal............................................ 4
3.3 Enzyme Purification......................................... 6
3.4 Blood Protein Purification ............................... 13
Section 4 Storage Conditions................................. 14
Section 5 References............................................... 14
Section 6 Product Information.............................. 17
Section 7 Technical Information........................... 18
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Section 1
Introduction
Affi-Gel blue affinity gel is a beaded, crosslinked
agarose gel with covalently attached Cibacron
F3GA dye. It contains ≥1.9 mg dye per ml of gel, and
has a capacity for albumin binding of greater than 11
mg/ml. Affi-Gel blue gel purifies a large range of
proteins from widely divergent origins. The blue dye
functions as an ionic, hydrophobic, aromatic, or
sterically active binding site in various applications.
Proteins that interact with Affi-Gel blue gel can be
bound or released with a high degree of specificity by
manipulating the composition of the eluant buffers. In
many cases, one can also predict what will interact with
the matrix and the general conditions under which
binding and elution will occur.
1
®
Blue
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Fig. 1. Cibacron blue coupled to agarose.
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
Affi-Gel blue gel is supplied ready to use as an
aqueous slurry of fully hydrated gel. It is available in
two convenient particle sizes: a faster flowing 50-100
mesh (150-300 µm) and a slower flowing, higher
capacity 100-200 mesh (75-150 µm). The gel is also
available in convenient Econo-Pac
can be used with the Econo System, FPLC
systems.
2
®
cartridges which
®
, and HPLC
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Section 2
Product Description
Matrix Bio-Gel A-5m agarose gel
Particle Sizes 150-300 µm (50-100 mesh)
Shipping Medium 0.05% NaN
Functional Group Cibacron blue
Typical Flow Rate* 15-25 cm/hr
Pressure limit 15 psi
Serum Capacity 0.2 ml/ml gel
Typical Albumin
Capacity 11 mg/ml
Stability
pH 4-10
Organic Solvents alcohols
Temperature 4-30 °C
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
80-150 µm (100-200 mesh)
3
or other preservative
3
3
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Section 3
Instructions for Use
3.1 Sample Preparation
Proper adjustment of the sample pH and ionic
strength is critical for consistent and reproducible results
when using dye affinity gels. The sample must be
exchanged into the appropriate application buffer. This
can be achieved by exchanging it into the application
buffer using Econo-Pac 10DG desalting columns,
Bio-Gel P-6DG desalting gel, or the Econo-Pac P6
cartridge. The choice of product depends on the sample
volume. Alternatively, the sample can be dialyzed
against the application buffer. All samples should be
filtered through a 0.45 µm filter.
3.2 Albumin Removal
Affi-Gel blue gel provides a simple first step in the
purification of many serum proteins by removing the
major serum constituent, albumin. The binding of albumin
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is so strong that a high concentration of salt or chaotropic
reagent is required to desorb the albumin. Other serum
proteins either do not bind to Affi-Gel blue gel or can be
eluted with relatively low concentrations of salt.
Table 1. Buffer Formulations For Albumin
Removal Procedure
A Buffer 20 mM phosphate buffer, pH 7.1
B Buffer 1.4 M NaCl, in 20 mM phosphate buffer,
pH 7.1
C Buffer 2 M guanidine HCl in 20 mM phosphate
buffer, pH 7.1
or 1.5 M NaSCN in 20 mM phosphate buffer,
pH 7.1
Procedure
1. Prepare a column of Affi-Gel blue gel, 50-100 mesh,
with a total bed volume of 5 ml of gel per milliliter
of serum to be processed.
2. Prewash the column with 2 bed volumes of buffer A.
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3. Equilibrate the serum sample in buffer A by
dialyzing overnight, or by rapid column desalting in
a column of Bio-Gel P-6 DG gel, on an Econo-Pac
10DG column, or on an Econo-Pac P6 cartridge.
4. Apply the equilibrated serum sample to the column.
5. Wash the column with 2 bed volumes of buffer A.
The effluent from this step contains the serum
proteins minus most of the albumin.
6. Optional step: elute the albumin with buffer B.
7. Whether or not the albumin was eluted, regenerate
the column with 2 bed volumes of buffer C.
3.3 Enzyme Purification
Affi-Gel blue gel has been used to purify a number
of enzymes. It has been particularly useful in the
purification of kinases, dehydrogenases, and other
nucleotide-dependent enzymes. The degree of
purification obtained with Affi-Gel blue gel is typically
much greater than that obtained using biospecific
affinity chromatography. It has been suggested that
enzymes containing a “dinucleotide fold” bind
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biospecifically to the Cibacron Blue F3GA dye.1In
many cases, the enzyme can be eluted from the Affi-Gel
blue gel with a specific nucleotide cofactor. Often, salt
is more effective for elution, indicating that other
mechanisms are sometimes involved.
2
Table 2 lists some applications in which Affi-Gel
blue gel has been used for enzyme purification.
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Table 2. Affi-Gel Blue Gel Enzyme Applications
Enzyme Source Eluant Reference
1-aminocyclopropane- Tomato pericarp tissue 50 mM KPO
1-carboxylate synthase 5 µM pyridoxal phosphate
3',5' cyclic AMP Dictyostelium 0.18 M NaCl 3
phosphodiesterase discoideum
3'-PGA diesterase Yeast 0.6 M KCl in 50 mM Tris-HCl 35
5-methyl-L-tetrahydrofolate Bovine liver 0.4-3.0 M KCl 6
reductase
Acylation stimulating protein Human plasma 3 M NaCl, 0.02 M phosphate 37
Adenylate cyclase Bovine brain 1 mM KCl, 8 mM ATP, 24
Alkaline phosphatase E. coli -39
Alkyl hydroperoxide reductase S. thphimurium 1.0 M KCl, 0.5 M NaCl 36
ATP:AMP phosphotransferase Bovine heart 0.4-2.0 M NaCl 10
Calmodulin-dependent cyclic Bovine brain 0.15-1.5 M NaCl 23
nucleotide phosphodiesterase
Carbamyl phosphate synthetase Frog liver 1 mM dithiothreitol 14
DNA polymerase Calf thymus 0-0.5 M KCl 12
Formamidopyrimidine-DNA E. coli 0.1-0.8 M KCl 4
glycosylase
Glutamate dehydrohenase Yeast 10 mM NADH, 1 M NH4Cl 15
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Eluant Reference
, 41
5 µM pyridoxal phosphate
4
0.18 M NaCl 3
0.6 M KCl in 50 mM Tris-HCl 35
0.4-3.0 M KCl 6
3 M NaCl, 0.02 M phosphate 37
1 mM KCl, 8 mM ATP, 24
16 mM MgCl
, 1 mM EDTA
2
-39
1.0 M KCl, 0.5 M NaCl 36
0.4-2.0 M NaCl 10
0.15-1.5 M NaCl 23
1 mM dithiothreitol 14
0-0.5 M KCl 12
0.1-0.8 M KCl 4
10 mM NADH, 1 M NH4Cl 15
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Table 2. Affi-Gel Blue Gel Enzyme Applications
(continued)
Enzyme Source Eluant Reference
Glutamine synthetase Salmonella typhimurium 5 mM ATP 8
Glyoxalase II Rat erythrocytes 0-0.2 M KCl 19
GMP reductase Human erythrocytes 1 mM NADPH, 2 mM GMP 21
GTP:RNA guanylyltransferase Wheat germ 0.05-0.75 M NaCl 5
Isocitrate dehydrogenase E. coli 2 mM NADP 16
Isocitrate dehydrogenase E. coli 2 mM NADP+ 13
MB creatine kinase Human heart 0.25 M NaCl 7
Membrane-bound Rat brain Triton X-100, NaCl, glycerol 38
phosphatidylinositol kinase
Methylenetetrahydrofolate Porcine liver 0-10 mM NADPH 17
reductase
Metmyoglobin reductase Bovine heart 1.0 M NaCl, 1 mM NADP+ 11
Phosphodiesterase Bovine brain 0.2 M NaCl 25
Replication protein A (RP-A) Animal cells - 40
RNA ligase E. coli 0.2 M NaCl, 2 mM ATP 20
Serine transhydroxymethylase Porcine liver 0.5 M KCl 18
Thymidylate Saccharomyces cervisiae 0.25-1 M KCl 22
Tyrosine phenollyase Erwinia herbicola 1.5 M NaCl, 0.5 mM mercaptoethanol 9
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Eluant Reference
0-0.2 M KCl 19
0.05-0.75 M NaCl 5
2 mM NADP 16
2 mM NADP+ 13
0.25 M NaCl 7
Triton X-100, NaCl, glycerol 38
0-10 mM NADPH 17
1.0 M NaCl, 1 mM NADP+ 11
0.2 M NaCl 25
-40
0.2 M NaCl, 2 mM ATP 20
0.5 M KCl 18
1.5 M NaCl, 0.5 mM mercaptoethanol 9
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Suggested Procedure for Enzyme Purification
1. Prepare a column of Affi-Gel blue gel, 100-200 mesh.
A 5 ml bed volume for every 20 mg of protein to be
chromatographed should be sufficient. Equilibrate the
column with starting buffer. The starting buffer should
be of low ionic strength, 50 mM or less. (Published
methods have used pH values from 6.0 to 8.5)
2. Dialyze sample against starting buffer. Alternatively,
the sample can be rapidly desalted in a column of
Bio-Gel P-6 DG gel, on an Econo-Pac 10DG
column, or on an Econo-Pac P6 cartridge.
3. Apply the sample to the column.
4. Wash the column with 2 bed volumes of starting
buffer.
5. Check the effluent for enzyme activity. If it is not
bound, then alter conditions. Change the pH,
decrease the ionic strength, or change the buffer.
6. The column may be eluted with a salt gradient or
with a competitive eluant such as a cofactor. Table 2
contains examples of eluants used in Affi-Gel blue
gel chromatography.
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7. Regenerate the column by washing it with 2 bed
volumes of 2 M guanidine HCl or 1.5 M NaSCN,
followed by 2 bed volumes of the starting buffer.
3.4 Purification of Blood Proteins
Affi-Gel blue gel has been used to separate and purify
a number of different serum and plasma proteins. Table 3
lists several examples. Gianazza and Arnaud
developed a single step method for general fractionation
of plasma proteins. By altering pH and ionic strength,
twenty-seven plasma proteins were separated, providing
better initial purification of individual proteins than many
of the two and three step methods commonly used.
Table 3. Purification of Blood Proteins on
Affi-Gel Blue Gel
Application Reference
Purification of human serum complement 26
Purification of rat alpha fetoprotein 27
Purification of rat serum albumin 28
Separation and purification of plasma proteins 29,30
Purification of alpha-2-macroglobulin 31
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29,30
have
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Section 4
Storage Conditions
The Affi-Gel blue gel should be stored at 4 °C in
0.02% sodium azide.
Section 5
References
1. Thompson, S. T., et al., Proc. Nat. Acad. Sci. USA, 72, 669
(1975).
2. Wilson, J. E., Biochem. Biophys. Res. Comm., 72, 816 (1973).
3. Dicou, E. and Brachet, P., Biochem. Biophys. Res. Comm.,
102, 1172 (1981).
4. Chetsanga, C. J., et al., Biochemistry, 20, 5201 (1981).
5. Keith, J. M., et al., Biochemistry, 21, 321 (1982).
6. Kattchee, P. A. and Guynn, R. W., Anal. Biochem., 118, 85
(1981).
7. Herman, C. A. and Roberts, R., Anal. Biochem., 106, 211
(1980).
8. Miller, E. S. and Brenchley, J. E., J. Biol. Chem., 256,
11307 (1981).
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9. Meadows, G. G. and Cantwell, G. S., Res. Comm. in
Chemical Pathology and Pharmacology, 30, 535 (1980).
10. Tomasselli, A. G. and Noda, L. H., Eur. J. Biochem., 103,
481 (1980).
11. Hagler, L., et al., J. Biol. Chem., 254, 6505 (1979).
12. Steinberg, J. A., et al., Cancer Research, 39, 4330 (1979).
13. Garnak, M. and Reeves, H. C., J. Biol. Chem., 254, 7915
(1979).
14. Mori, M. and Cohen, P. P., J. Biol. Chem., 253, 8337 (1978).
15. Hemmings, B. A., J. Biol. Chem., 253, 5255 (1978).
16. Vasquez, B. and Reeves, H. C., Biochem. Biophys. Acta,
578, 31 (1979).
17. Mathews, R. G. and Haywood, B. J., Biochemistry, 18,
4845 (1979).
18. Braman, J. C., et al., Preparative Biochemistry, 11, 23 (1981).
19. Ball, J. C. and Vander Jagt, D. L., Anal. Biochem., 98, 462
(1979).
20. McCoy, M. I. M., et al., Biochem. Biophys. Acta, 562, 149
(1979).
21. Spector, T., et al., J. Biol. Chem., 254, 2308 (1979).
22. Bisson, L. F. and Thorner, J., J. Biol. Chem., 256, 12456
(1981).
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23. Sharma, R. K., et al., J. Biol. Chem., 255, 5916 (1980).
24. Wescott, K. R., et al., Proc. Nat. Acad. Sci. USA, 76, 204
(1979).
25. Wallace, R. W., et al., J. Biol. Chem., 254, 377 (1979).
26. Gee, A. P., et al., J. Immunol. Methods, 30, 119 (1979).
27. Miyazaki, M., et al., Acta. Med. Okayama, 35, 427 (1981).
28. Day, J. F., et al., J. Biol. Chem., 254, 9394 (1979).
29. Gianazza, E. and Arnaud, P., Biochem. J., 201, 129 (1982).
30. Gianazza, E. and Arnaud, P., Biochem. J., 203, 637 (1982).
31. Arnaud, P. and Gianazza, E., FEBS Letters, 137, 157 (1982).
32. Ledden, D. J., et al., Biochem. J. (1982).
33. Burgett, M. W. and Greenley, L. V., Am. Lab. (1977).
34. Papp, S., et al., Anal. Biochem., 154, 327 (1986).
35. Johnson, A. W. and Demple, B., J. Biol. Chem., 263, 18009
(1988).
36. Jacobson, F. S., et al., J. Biol. Chem., 264, 1488 (1989).
37. Cianflone, K. M., et al., J. Biol. Chem., 264, 426 (1989).
38. Yamakawa, A. and Takenawa, T., J. Biol. Chem., 263,
17555 (1988).
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39. Butler-Ransohoff, J. E., et al., Proc. Nat. Acad. Sci. USA,
85, 4276 (1988).
40. Wold, M. W. and Kely, T., Proc. Nat. Acad. Sci. USA, 85,
2523 (1988).
41. Bleeker, A. B., et al., Proc. Nat. Acad. Sci. USA, 83, 7755
(1986).
Section 6
Product Information
Catalog
Number Product Description
153-7301 Affi-Gel Blue Gel, 50-100 mesh, 100 ml
153-7302 Affi-Gel Blue Gel, 100-200 mesh, 100 ml
732-0101 Econo-Pac Blue Cartridge, 1 x 5 ml
732-0105 Econo-Pac Blue Cartridge, 5 x 5 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, 1 x 5 ml
732-0015 Econo-Pac P6 Cartridge, 5 x 5 ml
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Section 7
Technical Information
For additional information and technical assistance,
contact your local Bio-Rad representative or call
Technical Service at 1-800-4BIORAD.
FPLC is a trademark of Pharmacia Biotech AB.
Cibacron is a trademark of Ciba Geigy.
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Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
LIT-590 Rev B
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