Bio-Rad Affi-Gel 15 Gel User Manual
Activated Immunoaff inity
Supports
Catalog Numbers
153-6046 Affi-Gel®10 Gel
153-6052 Affi-Gel 15 Gel
153-6098 Affi-Gel 10 and 15 Gel
Table of Contents
Section 1 Introduction...................................... 1
Section 2 Coupling Chemistry......................... 2
Section 3 General Coupling Conditions......... 5
3.1 pH Dependence......................................... 5
3.2 Temperature .............................................. 12
3.3 Time .......................................................... 12
3.4 Ligand Concentration................................ 12
Section 4 Recommended Storage Conditions 16
Section 5 General Instructions........................ 16
5.1 Aqueous Coupling..................................... 16
5.2 Anhydrous Coupling................................. 19
Section 6 Monitoring For Protein Coupling.. 22
Section 7 Troubleshooting............................... 22
Section 8 Using the Coupled Support............. 25
i
Section 9 Immunoaffinity Chromatography
with Affinity Supports..................... 26
9.1 Adsorption of the Sample.......................... 26
9.2 Removal of Unbound Solutes.................... 29
9.3 Elution Strategies ...................................... 30
9.4 Special Considerations for Labile Antigens34
9.5 Renaturation of Eluted Proteins ................ 35
Section 10 Ordering Information ..................... 36
Section 11 References......................................... 38
Section 1
Introduction
Affi-Gel 10 and Affi-Gel 15 affinity supports are activated immunoaffinity supports that offer rapid, high efficiency coupling for all ligands with a primary amino
group, including proteins throughout the entire range of
pIs and low molecular weight compounds such as peptides.1 Both Affi-Gel 10 and 15 supports are N-hydroxysuccinimide esters of a derivatized crosslinked agarose gel
bead support, and both couple to ligands spontaneously in
aqueous or non-aqueous solution.
The Affi-Gel 10 support, which contains a neutral 10atom spacer arm, has been used to couple a variety of
materials in affinity chromatography, immunoadsorption,
and other techniques. The Affi-Gel 15 support contains a
cationic charge in its 15-atom spacer arm which significantly enhances coupling efficiency for acidic proteins at
ii
1
C-ON
O
O
O
+ R-NH
2
pH 6.5 to 8.5
Buffer
C-N-R
O
O
O
H
+ HO-N
physiological pH. Both Affi-Gel 10 and Affi-Gel 15 supports offer the following advantages:
• Covalent amide bonds couple the protein to the terminal carboxyl of the spacer arm
• Highly stable in chaotropic agents and from pH 2-11
• Rapid, gentle coupling within 4 hours
• Easy to use
• High capacity of up to 35 mg protein per ml
Section 2
Coupling Chemistry
Ligands with free alkyl or aryl amino groups will cou-
ple spontaneously with Affi-Gel 10 or 15 supports in
aqueous or non-aqueous solution (refer to Figure 1). Upon
addition of ligand, the N-hydroxysuccinimide is displaced, and a stable amide bond is formed. Since the reac-
2
tive ester immobilized on the gel is highly selective for
primary amino groups, spurious side reactions with the
ligand (i.e., cross-linking or other modifications in free
solution) are eliminated. Free sulfhydryls are among
functional groups other than primary amines known to
compete for coupling.
Affi-Gel 10 and Affi-Gel 15 supports are well suited
for coupling low molecular weight ligands. This can be
done in aqueous solution or, when solubility of the ligand
permits, in organic solvent.
Fig. 1. Coupling reaction of Affi-Gel supports with ligand
containing free amino groups.
3
Product Description for Affi-Gel 10 and 15 gels
OCH2CONH(CH2)3N(CH2)3NHCO(CH2)2COON
O
O
CH
3
H
+
OCH2CONH(CH2)2NHCO(CH2)2COON
O
O
Matrix Bio-Gel A-5m agarose gel
Exclusion limit (M
Bead size 75-300 µm (50-200 mesh)
Spacer arm
Affi-Gel 10
Affi-Gel 15
Shipping medium 100% isopropanol
Capacity
Chemical capacity 15 µmoles/ml of gel
Protein capacity 35 mg/ml
Stability of unreacted support
Temperature -70 to 0 °C
pH range 3-10
Organic solvents stable in alcohols, DMSO, dioxane, formamide
Storage -20 °C 1 year
) 5,000,000
r
-70 °C 1.5 years
4
Section 3
General Coupling Conditions
3.1 pH Dependence
A major advantage of Affi-Gel 10 and 15 supports is
the mild conditions which will permit coupling. This is
particularly advantageous in applications which involve
sensitive enzymes or other proteins that irreversibly lose
biological activity when exposed to conditions outside
their physiological range. Coupling can be achieved with
Affi-Gel 10 and 15 supports between pH 3.0 to 10.0.
In order to maintain pH control, a minimum buffer
strength of 10 millimolar is recommended. Suitable
buffers include MES, MOPS, HEPES, POPSO, acetate,
and bicarbonate. Do not use buffers such as Tris or
glycine. They contain primary amino groups which will
couple to the gel, as will any primary amine-containing
compound which contaminates the ligand preparation.
5
The Affi-Gel 10 support couples proteins best at a pH
near or below their isoelectric point, and the Affi-Gel 15
support couples proteins best near or above their isoelectric point. Therefore, when coupling at neutral pH (6.5-
7.5), the Affi-Gel 10 support is recommended for proteins
with isoelectric points of 6.5 to 11 (neutral or basic proteins), and the Affi-Gel 15 support is recommended for
proteins with isoelectric points below 6.5 (acidic proteins). See Table 1.
The difference in coupling efficiency of the Affi-Gel
10 and Affi-Gel 15 supports for acidic and basic proteins
can be attributed to interactions between the charge on the
protein and charge on the gel. Hydrolysis of some of the
active esters during aqueous coupling will impart a slight
negative charge to the Affi-Gel 10 support. This negative
charge will attract positively charged proteins (proteins
buffered at a pH below their isoelectric point) and enhance
their coupling efficiency. Conversely, the negative charge
will repel negatively charged proteins (proteins buffered
at a pH above their isoelectric point) and lower their coupling efficiency. The Affi-Gel 15 support, due to the tertiary amine incorporated into its arm, has a slight overall
positive charge, and the effects are reversed.
6
7
Fig. 2. Protein coupling with Affi-Gel and Affi-Gel 15 sup-
0
10
20
30
40
50
60
70
80
90
100
% Protein Coupled
0
5
10
15
20
mg coupled
ml gel
34567891011
pl
ports. Coupling conditions: Each protein solution (3 ml 0.1 M
MOPS, pH 7.5, containing 40 mg protein) was combined with
2 ml of Affi-Gel media. The gel slurry was mixed at 4 °C for 2
hours, and then stripped with 7 M urea containing 1 M NaCl.
The uncoupled protein was determined, using published
1cm
E
, by dilution of an aliquot of the urea effluent into 0.1 M
280
HCl and measurement of the absorbance at 280 nm (
Affi-Gel 15 gel; ●●—●● Affi-Gel 10 gel).
8
●—●
Table 1. Protein Coupling to Affi-Gel 10 and
Affi-Gel 15 Support
Protein pl Affi-Gel 15 Gel Affi-Gel 10 Gel
1. Fetuin 3.3 43 3.0
2. Alpha-1-antitrypsin 4.0 76 5.0
3. Ovalbumin 4.7 70 8.5
4. Bovine serum albumin 4.9 80 14
5. Human transferrin 5.9 87 36
6. Bovine hemoglobin 6.8 59 83
7. Human globulin 5.8-7.3 39 90
8. Myoglobin 6.8-7.8 10 85
9. Cytochrome c 9.3 0 90
10. Lysozyme 10-11 1 95
Coupling Efficiency (%)
9
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