Bio-Rad ProteoMiner Protein Enrichment Kits - Manual (Page 16)

centrifuge at 1,000 x g for 30–60 sec. Discard collected material.
4. Repeat steps 2 and 3 two more times.
Step 4 – Sequential Elution
(Reagents required for this step are included with the ProteoMiner sequential
elution reagents, catalog #163-3003.)
When using the ProteoMiner sequential elution kit, the elution reagent and
rehydration reagent supplied with the ProteoMiner kit are not needed. Instead
use elution reagents 1–4 supplied with the ProteoMiner sequential elution
reagents (catalog #163-3003).
1. Carefully add 200 µl wash buffer on all sides of the column to ensure
none of the beads are stuck to the sides of the column.
2. Centrifuge at 1,000 x g for 30–60 sec. Discard collected material. If using
vacuum up to this point, you will now need to switch to centrifugation.
3. Attach bottom cap to the column (take caution to ensure the bottom cap
is tightly attached). Add 20 µl of elution reagent 1 to spin column.
Incubate at room temperature and lightly vortex several times over a
period of 10 min.
4. Remove bottom cap, place column in collection tube labeled F1 and
centrifuge at 1,000 x g for 30–60 sec to collect the elution. This elution
contains your eluted proteins. Do not discard.
5. Repeat steps 3 and 4 two more times and collect both elutions in tube F1.
6. Attach bottom cap to column and add 20 µl of elution reagent 2 to
column. Incubate at room temperature and lightly vortex several times
over a period of 10 min.
7. Remove bottom cap, place column in collection tube labeled F2 and
centrifuge at 1,000 x g for 30–60 sec to collect the elution. This elution
contains your eluted proteins. Do not discard.
8. Repeat steps 6 and 7 two more times and collect both eluents in tube F2.
(If treating plasma samples, a white precipitate will form when adding
elution reagent 2. Before using eluent in downstream applications, spin
and use supernatant).
9. Attach bottom cap to column, place column in collection tube labeled F3
and add 20 µl of elution reagent 3 to column. Incubate at room
temperature, and lightly vortex several times over a period of 10 min.
10. Centrifuge at 1,000 x g for 30–60 sec to collect the elution. This elution
contains your eluted proteins. Do not discard.
11. Repeat steps 9 and 10 two more times and collect both elutions in tube F3.
12. Attach bottom cap to column and add 20 µl of elution reagent 4 to
column. Incubate at room temperature and lightly vortex several times
over a period of 5 min.
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