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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
3.2
3.3
3.4

Sample Preparation .......................................... 4
Albumin Removal............................................ 4
Enzyme Purification......................................... 6
Blood Protein Purification ............................... 13

Section 4

Storage Conditions................................. 14

Section 5References............................................... 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® BlueF3GA dye. It contains ≥1.9 mg dye per ml of gel, andhas a capacity for albumin binding of greater than 11
mg/ml. Affi-Gel blue gel purifies a large range ofproteins from widely divergent origins. The blue dyefunctions as an ionic, hydrophobic, aromatic, or
sterically active binding site in various applications.
Proteins that interact with Affi-Gel blue gel can bebound 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 withthe matrix and the general conditions under whichbinding and elution will occur.

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NH2

R1
R2
NHoN
NH

NNH
N
SO2ONaR1 = H or SO2ONa
R2 = SO2ONa or HO
CrosslinkedAgaroseFig. 1. Cibacron blue coupled to agarose.

Affi-Gel blue gel is supplied ready to use as anaqueous slurry of fully hydrated gel. It is available intwo convenient particle sizes: a faster flowing 50-100
mesh (150-300 µ m) and a slower flowing, higher
capacity 100-200 mesh (75-150 µm). The gel is alsoavailable in convenient Econo-Pac ® cartridges which
can be used with the Econo System, FPLC®, and HPLC
systems.

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Section 2
Product Description
MatrixParticle Sizes
Shipping Medium
Functional Group
Typical Flow Rate*
Pressure limit
Serum CapacityTypical Albumin
Capacity
Stability
pH
Organic Solvents
Temperature
Storage*

Bio-Gel A-5m agarose gel
150-300 µm (50-100 mesh)
80-150 µm (100-200 mesh)
0.05% NaN3
Cibacron blue
15-25 cm/hr
15 psi0.2 ml/ml gel
11 mg/ml
4-10alcohols
4-30 °C
1 year at 4 °C, in 0.02% NaN3
or other preservative

Flow rate determined using a 1.5 x 20 cm column, and ahydrostatic pressure of 1:1
3

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Instructions for Use
3.1 Sample Preparation
Proper adjustment of the sample pH and ionicstrength is critical for consistent and reproducible results
when using dye affinity gels. The sample must be
exchanged into the appropriate application buffer. Thiscan be achieved by exchanging it into the applicationbuffer 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 samplevolume. 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 thepurification of many serum proteins by removing themajor 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 beeluted 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 phosphatebuffer, pH 7.1or

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 milliliterof 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 ina 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 numberof enzymes. It has been particularly useful in thepurification of kinases, dehydrogenases, and othernucleotide-dependent enzymes. The degree ofpurification obtained with Affi-Gel blue gel is typicallymuch greater than that obtained using biospecificaffinity chromatography. It has been suggested thatenzymes containing a “dinucleotide fold” bind6

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biospecifically to the Cibacron Blue F3GA dye. 1 In
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 othermechanisms 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
EnzymeSourceEluant1-aminocyclopropane1-carboxylate synthase
3',5' cyclic AMP
phosphodiesterase
3'-PGA diesterase
5-methyl-L-tetrahydrofolate
reductase
Acylation stimulating protein
Adenylate cyclase

Tomato pericarp tissue

50 mM KPO4,5 µM pyridoxal phosphate
0.18 M NaCl

410.6 M KCl in 50 mM Tris-HCl0.4-3.0 M KCl

3563724

E. coliS. thphimurium
Bovine heart
Bovine brain

3 M NaCl, 0.02 M phosphate
1 mM KCl, 8 mM ATP,
16 mM MgCl2, 1 mM EDTA
1.0 M KCl, 0.5 M NaCl
0.4-2.0 M NaCl
0.15-1.5 M NaCl

39361023

Frog liver
Calf thymus
E. coli

1 mM dithiothreitol
0-0.5 M KCl
0.1-0.8 M KCl

14124Yeast

10 mM NADH, 1 M NH4Cl

15

Alkaline phosphatase
Alkyl hydroperoxide reductase
ATP:AMP phosphotransferase
Calmodulin-dependent cyclicnucleotide phosphodiesterase
Carbamyl phosphate synthetase
DNA polymerase
Formamidopyrimidine-DNAglycosylase
Glutamate dehydrohenase
8Dictyostelium
discoideum
Yeast
Bovine liver
Human plasma
Bovine brain

Reference3LIT590B

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Page 9nsEluant
sue

Reference

50 mM KPO4,
5 µM pyridoxal phosphate
0.18 M NaCl

41

0.6 M KCl in 50 mM Tris-HCl
0.4-3.0 M KCl

35
6

3 M NaCl, 0.02 M phosphate
1 mM KCl, 8 mM ATP,
16 mM MgCl2, 1 mM EDTA
1.0 M KCl, 0.5 M NaCl
0.4-2.0 M NaCl
0.15-1.5 M NaCl

3724393610231 mM dithiothreitol
0-0.5 M KCl
0.1-0.8 M KCl

1412410 mM NADH, 1 M NH4Cl

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Table 2. Affi-Gel Blue Gel Enzyme Applications(continued)
EnzymeSourceEluantGlutamine synthetase
Glyoxalase II
GMP reductase
GTP:RNA guanylyltransferase
Isocitrate dehydrogenase
Isocitrate dehydrogenase
MB creatine kinase
Membrane-boundphosphatidylinositol kinase
Methylenetetrahydrofolatereductase
Metmyoglobin reductase
Phosphodiesterase
Replication protein A (RP-A)
RNA ligase
Serine transhydroxymethylase
Thymidylate
Tyrosine phenollyase

Salmonella typhimurium
Rat erythrocytes
Human erythrocytes
Wheat germ
E. coliE. coli
Human heart
Rat brain

5 mM ATP0-0.2 M KCl
1 mM NADPH, 2 mM GMP
0.05-0.75 M NaCl
2 mM NADP
2 mM NADP+
0.25 M NaCl
Triton X-100, NaCl, glycerol

81921
5
16
13
738

Porcine liver

0-10 mM NADPH

17

Bovine heart
Bovine brain
Animal cells
E. coli
Porcine liver
Saccharomyces cervisiae
Erwinia herbicola

1.0 M NaCl, 1 mM NADP+
0.2 M NaCl
0.2 M NaCl, 2 mM ATP
0.5 M KCl
0.25-1 M KCl
1.5 M NaCl, 0.5 mM mercaptoethanol

112540201822910ReferenceLIT590B

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nsEluant
uriumsvisiae

Reference

5 mM ATP0-0.2 M KCl
1 mM NADPH, 2 mM GMP
0.05-0.75 M NaCl
2 mM NADP
2 mM NADP+
0.25 M NaCl
Triton X-100, NaCl, glycerol

81921516137380-10 mM NADPH

17

1.0 M NaCl, 1 mM NADP+
0.2 M NaCl
0.2 M NaCl, 2 mM ATP
0.5 M KCl
0.25-1 M KCl
1.5 M NaCl, 0.5 mM mercaptoethanol

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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 thecolumn with starting buffer. The starting buffer should
be of low ionic strength, 50 mM or less. (Publishedmethods 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 ofBio-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 notbound, then alter conditions. Change the pH,
decrease the ionic strength, or change the buffer.
6. The column may be eluted with a salt gradient orwith a competitive eluant such as a cofactor. Table 2
contains examples of eluants used in Affi-Gel blue
gel chromatography.
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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 ProteinsAffi-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 Arnaud29,30 havedeveloped a single step method for general fractionationof plasma proteins. By altering pH and ionic strength,
twenty-seven plasma proteins were separated, providing
better initial purification of individual proteins than manyof 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
3113

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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 5References
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).
14

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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
NumberProduct 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 ml732-0105

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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9/3/98 1:30 PMPage 18Section 7
Technical Information
For additional information and technical assistance,
contact your local Bio-Rad representative or callTechnical 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