Bio-Rad ProteoMiner Protein Enrichment Kits - Manual (Page 15)

Note: Kit contains one capless collection tube per spin column for the
following steps: column preparation, sample binding, and sample wash.
Kit contains one capped collection tube per spin column to be used for
the elution step, allowing for easy storage of your eluted sample.
3. Replace the bottom cap and add 200 µl wash buffer, then replace top cap.
4. Rotate column end-to-end several times over a 5 min period.
5. Remove bottom cap, place the column in a capless collection tube and
centrifuge at 1,000 x g for 30–60 sec to remove water. Discard collected
material.
6. Repeat steps 3 and 4.
7. Remove bottom cap, place the column in a capless collection tube and
centrifuge at 1,000 x g for 30–60 sec to remove the wash buffer. Discard
collected material.
8. Replace bottom cap on spin column. The column now contains 20 µl of
settled beads and is ready for sample binding.
Step 2 – Sample Binding
Samples should be free of precipitate. Centrifuge samples at 10,000 x g for
10 min to clarify. Take precautions to avoid the bottom aggregate proteins and
top lipid layer when recovering your sample. It is recommended that at least
200 µl of sample (protein concentration >50 mg/ml) is added to the column,
lower volumes may not achieve optimal results. For other sample types,
please refer to Section 5: Sample Considerations.
If using plasma: Add 180 µl plasma (nonheparinized) and 20 µl plasma
preparation buffer to spin column. Replace top cap and rotate column end-to-end
for 2 hr at room temperature. After approximately 1 hr of binding you may see
clumping; this is expected and will not negatively impact your sample
preparation. Heparinized plasma is not compatible with this kit.
For all other sample types: Add 200 µl of sample to column and replace
top cap. Rotate column on a platform or rotational shaker for 2 hr at room
temperature.
Step 3 – Sample Wash
(Reagents required for this step are included in the ProteoMiner kit, catalog
#163-3006.)
1. Remove caps, place column in a capless collection tube and centrifuge at
1,000 x g for 30–60 sec. Discard collected material.
2. Replace the bottom cap and add 200 µl of wash buffer to column, then
replace top cap and rotate end-to-end several times over a 5 min period.
3. Remove bottom cap, place column in a capless collection tube and
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