Bio-Rad ProteoMiner Protein Enrichment Kits User Manual
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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. If needed, 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, as lower volumes may not achieve optimal
results. For other sample types, please refer to Section 5: Sample
Considerations.
1.
Add 200 µl of sample to column. Replace top cap and rotate column
on a platform or rotational shaker for 2 hr at room temperature.
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 200 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 two more times.
Step 4 – Elution
1.
After all wash buffer has been removed, replace the bottom cap and add
200 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 20 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.
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