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Dye Ligand Chromatography 65
6. Miscellaneous: Fraction tubes, chromatography columns (from GE, Millipore,
Omnifit or equivalent).
3. Method
3.1. Batch Binding
1. Obtain 50 ml of cell culture supernatant per dye resin to be tested. This should
be clarified down to 0.2μm by a combination of centrifugation, capsule filtration
and vacuum filtration (see Notes 2 and 4).
2. Ten milliliters of the cell culture supernatant is dialyzed against each binding
buffer (see “Instructions: Slyde-A-Lyzer Dialysis Products”). A stir plate will be
required to mix each of the dialysis containers at room temperature overnight
(see Note 5).
3. To prepare the dye resin for use, aliquot 10 ml of each binding buffer into a new
set of five labeled test tubes (one for each separate pH). Aliquot 50 ml of the dye
ligand resin into the appropriate test tube (taking into account the slurry factor;
for a 50% slurry transfer 100 ml). The slurry may be in the shipping solution
because the first step below will rinse the gel. This is an important step, so care
should be taken to aliquot the correct amount of resin.
a. Use the razor to cut the tip off of the micropipette to be used. This will insure
that the gel is not prevented from freely entering the micropipette.
b. Calculate the amount of slurry to be added: 50 ml × the total slurry
volume/settled resin volume. Pipette this amount into each polypropylene
tube.
c. Cap the test tubes and vortex.
4. Spin the resin down in a centrifuge (Gt = 10 6 s) and remove the buffer using a
serological pipette, without disturbing the gel. Do not decant; gel will inevitably
be lost. Use a serological pipette.
5. Add the dialyzed protein solution to the appropriate test tubes (pH 4 dialysate
with pH 4 binding buffer, etc.). If dialysis has resulted in a change of volume,
add the equivalent of 10 ml of starting cell culture supernatant (i.e., if the Slyde-
A-Lyzer contents swell from 10 to 13 ml, add the entire 13 ml). Mix overnight
at room temperature. Note that prior knowledge of protein stability may dictate
specific incubation conditions (temperature, incubation time, etc.) at this point.
6. Spin the resin down in a centrifuge (Gt = 10 6 s) and remove the supernatants
without disturbing the gel. Transfer the supernatants to labeled tubes. The supernatants,
containing unbound protein, should be stored prior to assay under conditions
favorable to target protein stability.
7. Add 5 ml of the appropriate elution buffer to each of the resin pellets. (The batch
binding experiments are carried out at constant pH, that is, use the same pH
binding and elution buffer conditions.). Mix for 10 min.
8. Spin the resin down in a centrifuge (Gt = 10 6 s) and remove the eluents using a
serological pipette as described above, label and store.
9. Assay the binding supernatants and the eluents for protein activity.