Waters Protein-Pak Epoxy-Activated Affinity Products - Manual (Page 4)

[ Care and Use ManUal ]
Protein-Pak Epoxy-Activated Affinity 4
The coupling supernatant and sodium chloride washes may be saved
to quantitate the unbound ligand.
e. Storage
Store the material containing immobilized ligand at 4 °C (do not
freeze) in a solution of:
– 0.01 to 0.1M sodium phosphate (pH 7.4) or
– 0.1 to 0.5 M sodium chloride or
– 0.01 % sodium azide
III. usIng the MICro-ColuMn
Prepare the Micro-Column containing the epoxy-activated packing
and isolate the selected product as follows.
a. Preparing the Micro-Column
1. Allow the Micro-Column to warm to room temperature.
2. Remove the cap at the bottom first and then the stopper at the
top. Removing the stopper and cap in this order prevents the dry
powder from scattering.
3. Place the Micro-Column in a ring stand or rack to allow test
tubes or beakers to be put underneath the cartridge.
4. If vacuum will be used to remove the liquid from the Micro-
Column, attach the vacuum source to the bottom of the Luer
outlet.
5. Wash the packing material in the Micro-Column by putting
1.5 mL of HPLC grade water in the reservoir and allow it to
flow through by gravity or vacuum.
6. Use the guidelines in Table 1, Section II to select coupling
buffer for your ligand. Take 1.5 mL of coupling buffer to wash
the packing material in the Micro-Column. Add the buffer to the
reservoir and allow it to flow through by the gravity or vacuum.
7. Put the cap on the bottom of the Micro-Column. Add an
additional 1.5 mL of coupling buffer, and stopper the top.
8. Place on a laboratory rotator for five minutes to thoroughly
equilibrate the material in the coupling buffer.
9. Remove the stopper and cap and let the buffer drain off by
gravity or vacuum. Put the cap back on the bottom of the
Micro-Column.
10. See Sections II, a and II, b for recommended coupling buffers and
conditions.
11. Add the ligand to be immobilized in 1.5 mL of the coupling
buffer (for optimum mixing).
12. Stopper the Micro-Column and place it on the laboratory
rotator at the desired temperature and time period. See
Sections II, a and II, e for recommended coupling, blocking,
washing, and storage conditions.
b. Isolation of the Selected Product
1. Wash the coupled packing with the elution buffer until the eluent
collected reaches steady or zero absorbance value at the desired
wavelength (use and UV-Visible spectrophotometer).
2. Wash the coupled packing with loading buffer at least 10
column volumes (5 mL).
3. Apply the crude, prefiltered sample (preferably in loading
buffer) to the Micro-Column.
4. Wash the unbound material off the Micro-Column until low
or zero absorbance is read for the eluent. Then wash the
Micro-Column with the elution buffer to low or zero absorbance.
5. Alternatively, the Micro-Column can be capped and the
biomolecule to be isolated can be applied to the column in
2.5 mL loading buffer; the Micro-Column can then be stoppered
and rotated for 10 minutes or longer to assure maximum
binding of the biomolecule to the ligand. After the stopper and
cap are removed, continue as outlined in Step 4 above.
6. Re-equilibrate in 5 mL of loading buffer if a second purification
is planned at this time.
7. While these Micro-Columns are recommended for single use
only, the Micro-Column may be reused at a later date; reuse is
dependent on the application, including degree of cleanliness
of packing material needed, stability of ligand and time elapsed
between uses.