Bio-Rad Aurum Ion Exchange Mini Kits User Manual
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14.
Place the column in a new 2.0 ml collection tube labeled “bound #2”.
15.
Wash with 0.3 ml of the Aurum AEX or CEX elution buffer. Centrifuge for
10 sec at 1,000 x g.
Sample Preparation for IEF Using Micro Bio-Spin 6 Chromatography
Desalting Procedure
1.
Invert the column sharply several times to resuspend the settled gel and
remove any bubbles. Snap off the tip and place the column in a 2.0 ml
collection tube. Remove the top cap. If the column does not begin to
flow, push the cap back on the column and remove it again to start the
flow. Allow the excess packing buffer to drain by gravity to the top of the
gel bed (about 2 min). Discard the drained buffer and place the column
back into the 2.0 ml tube.
2.
Centrifuge for 4 min in a microcentrifuge at 1,000 x g to remove the
residual packing buffer. Discard the buffer.
3.
Place the column in a clean 1.5 or 2.0 collection tube. Carefully apply the
sample (20–75 µl) directly onto the center of the column. Application of
more or less than the recommended sample volume may decrease
column performance.
4.
After loading sample, centrifuge the column for 4 min at 1,000 x g.
5.
Following centrifugation, the purified sample is now in the low ionic
strength binding buffer. Molecules smaller than the column’s exclusion
limit will be retained by the column.
6.
Dispose of the used column.
Buffer Exchange
The gel in the Micro Bio-Spin columns is suspended in Tris buffer, pH 7.4.
The gel matrix is compatible with most aqueous buffers. Buffer exchange can
be achieved using the following procedure.
1.
Follow steps 1 and 2 in the Desalting Procedure section.
2.
Apply the new buffer in 500 µl aliquots. After each application of new
buffer, let the buffer drain out by gravity, or centrifuge the column for
1 min to remove the buffer. Discard buffer from collection tube. Repeat as
required. Three washes result in >99% of the buffer exchanged. Four
washes result in >99.9% of buffer exchanged.
3.
Sample can now be applied to the column as directed in steps 3–6 in the
Desalting Procedure section.
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