Bio-Rad ProteoMiner Protein Enrichment Kits User Manual
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Section 7
Instructions for Use With ProteoMiner™
Small-Capacity Kits (Catalog #s 163-3006
and 163-3008)
This protocol has been optimized for plasma and serum samples with
protein concentrations of >50 mg/ml (requires total protein load >10 mg). For
other sample types, please refer to Section 5: Sample
Considerations.
Note: A ProteoMiner sequential elution kit (catalog #163-3010) is available for
researchers using SELDI or other downstream protein separation analysis
methods, other than 2-D gel electrophoresis, and who wish to access
additional proteins.
If using the ProteoMiner sequential elution kit, refer to page 12.
Step 1 – Column Preparation
Vacuum (at 16 mm Hg) can replace centrifugation for column preparation,
sample binding, and sample wash steps if desired. (Vacuum manifold is
available through Bio-Rad, catalog #732-6470.)
1.
First remove the top cap and then snap off the bottom cap from each of
the spin columns you will be using.
Note: Do not discard top or bottom caps, they will be reused
throughout the protocol. If beads settle in top cap, replace after removing
bottom plug and centrifuge with top cap on column. To use bottom cap
as a plug, invert and firmly place in bottom of spin column.
2.
Place the column in a capless collection tube and centrifuge at 1,000 x g
for 30–60 sec to remove the storage solution. Discard collected material.
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 buffer. Discard collected
material.
6.
Repeat steps 3 and 4.
7.
Remove caps, 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.
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