Bio-Rad ProteoMiner Protein Enrichment Kits - Manual (Page 14)

11. Repeat steps 9 and 10 two more times and collect both elutions in tube F3.
12. Attach bottom cap to column and add 100 µl of elution reagent 4 to
column. Incubate at room temperature and lightly vortex several times
over a period of 5 min.
13. Remove bottom cap, place column in collection tube labeled F4 and
centrifuge at 1,000 x g for 30–60 sec to collect the elution. This elution
contains your eluted proteins. Do not discard.
14. Repeat steps 12 and 13 two more times and collect both eluents in tube F4.
15. Store elutions at –20°C or proceed with downstream analysis. (See
Section 9 for more information on preparing sample for analysis.)
Section 9
Instructions for Use With Sequential
Elution Kit (Small-Capacity)
The ProteoMiner™ sequential elution small-capacity kit combines the
ProteoMiner kit (catalog #163-3006) and the ProteoMiner sequential elution
reagents (catalog #163-3003) and is available for researchers using SELDI or
other downstream protein separation analysis methods who wish to access
additional proteins. This kit is NOT compatible with 2-D gel electrophoresis.
This protocol has been optimized for plasma and serum samples with
protein concentration of >50 mg/ml (requires total protein load >10 mg). For
other sample types, please refer to Section 5: Sample
Considerations.
Step 1 – Column Preparation
(Reagents and columns required for this step are included in the ProteoMiner
kit, catalog #163-3006.)
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.
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