Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 15)

5. Apply 500 µl PBS, pH 7.4, to the top of the column and centrifuge at
1,000 x g for 2 min. Discard the buffer from the collection tube.
6. Repeat step 5 four additional times. On the last wash step, centrifuge
for 4 min.
7. Place the column in a clean and labeled 1.5 or 2 ml microcentrifuge
tube. Carefully apply the protein sample (30–100 µl) directly to the
center of the column.
Note: Application of more or less than the recommended sample
volume may decrease column performance.
8. Centrifuge the column for 5 min at 1,000 x g. The purified protein
sample is now in PBS, pH 7.4. Place the sample on ice and calculate
the amount of protein recovered using a Bio-Rad DC protein assay kit
or any other protein assay of choice.
Note: One coupling reaction requires 5–12 µg protein depending on
the protein used. If you are coupling for the first time, you may wish to
prepare multiple coupling reactions varying the amount of protein
used to determine the optimal coupling conditions for your protein.
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