Waters Protein-Pak Epoxy-Activated Affinity Products - Manual (Page 3)

[ Care and Use ManUal ]
Protein-Pak Epoxy-Activated Affinity 3
Table 1: Guidelines for Coupling to an Epoxy-Activated Packing
Material
* For insoluble or partially soluble ligands, an organic solvent may be
added to any of the above buffers.
To couple the ligand
1. Weigh out the amount of material required to pack the column.
The bulk density of the material is about 0.5 g/mL, so about
0.5 grams of material is required per mL of column volume.
2. Place the material in a tube or bottle with a screw cap that fits
securely. A screw cap is required so that no liquid is lost when
the container is rotated on a laboratory wheel or shaken.
3. Add three parts coupling buffer or solvent to one part packing
material, slurry or suspend, and rotate on a laboratory wheel or
shaker for 5 minutes. Let the material settle.
4. Decant the supernatant liquid and then wash the packing mate-
rial three times with fresh coupling buffer or solvent, using part
buffer or solvent, using three parts buffer or solvent to one part
packing material.
5. Dissolve the ligand in the coupling buffer or solvent in a test
tube. Keep the concentration of the ligand solution as high as
possible.
Note: During the coupling step, the final volume of buffer mass of
affinity material should be 3 to 4 mL solution per gram of material.
6. Following the guidelines shown in Table 1, mix the ligand
solution with the material using a rotating wheel. Alternatively,
when carrying out couplings at temperature above room tem-
perature, use a shaker in a water bath. Do not use a magnetic
stirrer because the silica beads may fragment or the ligand may
be damaged.
c. Blocking Procedure
1. The blocking solution may be added after the coupling solution
is removed or added directly to the reaction mixture. Block
unreacted epoxy groups with a final concentration of 1 M
ethanolamine solution at pH 9.5 in coupling buffer (2 mL/g) by
rotating for 18 to 24 hours at 4 °C.
2. Filter off the coupling/blocking supernatant using a coarse-
porosity, sinterd glass funnel. Save the solution to quantitate
the amount of unbound ligand.
d. Washing Procedure
Perform the following sequence of washes to remove excess ligand
and blocking solution. Use 6 mL of solution per gram of packing
material for each wash.
1. Coupling buffer or solvent (one wash)
Note: If coupling is performed in an organic solvent, initial washes
should be of an increasing polarity; for example toluene, acetone, and
then water.
2. 1 M NaCl solution (four washes).
3. Use one of the following buffers (one wash):
Loading buffer based on the application
Storage buffer (see Section II, e below)
Functional Group on the
Ligand
Coupling Conditions
Primary, secondary, and
aromatic amines and
sulfhydryl Any of the following at 20 °C to 55 °C, 17- 24
hours:
• 0.1-1.0M phosphate pH 6-7.5
• 0.1 M borate, pH 8- 9.5
• Organic solvents
Hydroxyl Any of of the following at 4 °C-45 °C, 17-72
hours:
• 0.1-1.0M phosphate pH 3-6.5
• Organic solvents such as dimethyl sulfoxide,
dimethyl formamide, acetonitrile
• Alkali pH 11-13 (no more than 18 hours)
Proteins including
enzymes and antibodies Any of of the following at 4 °C-45 °C, 24-72
hours:
• 0.1-1.0M phosphate pH 3-7.5
• 0.1-0.5M carbonate or borate, pH ≥
• 8.0 with or without 0.05 to 1.0 M NaCl