Bio-Rad ProteoMiner Protein Enrichment Kits User Manual
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Note: If using plasma, clumping may occur after 1 hr of binding; this is expected
and will not negatively impact your sample preparation. Heparinized plasma is not
compatible with this kit.
Step 3 – Sample Wash
1.
Remove bottom cap, 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 600 µl of wash buffer to column. Replace
top cap and rotate from end-to-end several times over a 5 min period.
3.
Remove bottom cap, place column in a capless collection tube and
centrifuge at 1,000 x g for 30–60 sec. Discard collected material.
4.
Repeat steps 2 and 3 three more times.
Step 4 – Elution
1.
After all wash buffer has been removed, replace the bottom cap and add
600 µl deionized water.
2.
Attach top cap and rotate end-to-end for 1 min.
3.
Remove caps, place column in a capless collection tube and centrifuge at
1,000 x g for 30–60 sec to remove water. Discard collected material.
If using vacuum up to this point, you will now need to switch to
centrifugation.
4.
Attach bottom cap to the column (take caution to ensure the bottom cap is
tightly attached). Add 100 µl of rehydrated elution reagent (refer to Section 4
for rehydration instructions) to the column and replace top cap. Lightly vortex
for 5 sec.
Note: For 2-D users who plan to use DIGE, this elution reagent will require clean
up and pH adjustment (described in Section 11). As an alternative you may elute
with DIGE buffer. However, this may result in a decreased yield and number of
protein spots.
5.
Incubate column at room temperature, lightly vortex several times over a
period of 15 min.
6.
Remove caps, place in a clean collection tube labeled E1 and centrifuge at
1,000 x g for 30–60 sec. This elution contains your eluted proteins; do not
discard.
7.
Repeat steps 5–6 two more times. (Elutions may be pooled or analyzed
individually. If analyzed individually, you will need additional collection tubes
not provided with kit.)
8.
Store elution at –20°C or proceed with downstream analysis. (For best
results, we recommend a clean up of your sample prior to analysis. See
Section 11 for more information on preparing sample for analysis.)
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