Bio-Rad Affi-Gel Blue Gel User Manual
Affi-Gel®Blue Gel
Instruction Manual
Catalog Numbers
153-7301
153-7302
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
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
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
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
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
4
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.
5