Waters Protein-Pak Epoxy-Activated Affinity Products - Manual (Page 5)

[ Care and Use ManUal ]
Protein-Pak Epoxy-Activated Affinity 5
c. Storage
– 0.01 to 0.1 M sodium phosphate (pH 7.4)
– 0.01 to 0.5 M sodium chloride
– 0.01% sodium azide
Iv. ChroMAtogrAphy guIdelInes
a. Column Packing
Pack the column with the affinity material containing an immobilized
ligand prepared as specified in the section above. One gram of
bulk material nominally provides 2.0 mL of column volume. The
bulk material may be slurry packed into any column. Following the
column packing guidelines from the care and use manual for the
column being used. The material can be slurry packed into an eluent
compatible with the ligand.
b. Operation
To perform an affinity chromatographic separation:
1. Prior to equilibrating with loading buffer, equilibrate the
column by washing with elution buffer until a steady baseline is
obtained. Then switch to a loading buffer for at least 5 column-
volumes.
2. Load the prefiltered sample containing the selected material
to be purified. See Steps 2 and 3 in Figure 1. When the affinity
interaction is weak or the kinetics are poor, reduce the flow rate
or recycle the sample though the column until a steady state
is achieved. The amount of solute remaining in the eluent will
reach a constant value.
3. Wash the column with loading buffer until a steady baseline is
obtained. See Step 4 in Figure 1.
4. Change the eluent to remove the bound sample. The limits on
the pH range and ionic strength of the elution buffer or solvent
are determined by the nature of the affinity ligand, the solute
and the column matrix for the specific application. See Step 5 in
Figure 1.
5. Re-equilibrate the column in the loading buffer.
V. STOrAGE AnD CArE OF ThE AFFInITy COLuMn
This section contains information on column life, storage, and
regeneration. Liquid chromatography columns have a finite life which
is directly related to the care and use they recieve. Store the column
under the conditions which prevent loss of ligand function. A general
rule is to store columns with protein ligands at 4 °C in pH 6 to 7.4
buffer containing a preservative such as 0.01% sodium azide. The
column cleaning procedure depends on the ligand and the applica-
tion. If removing debris, a typical procedure is to wash the columns
with 10 column volumes of the following solutions in the following
order:
1. Low pH wash, 5% to 10% acetic acid.
2. HPLC grade water or equivalent
3. 1 M sodium chloride
4. HPLC grade water or equivalent
5. Re-equilibrate the column in the chromatography startiing
buffer.