Bio-Rad ProteoMiner Protein Enrichment Kits - Manual (Page 17)

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 11 for more information on preparing sample for analysis.)
Section 10
Instructions for Use With Bulk Beads
Step 1 – Preparation
1. Before using the ProteoMiner™ beads (525 mg), swell the beads by
rehydrating with 10 ml, 20% v/v aqueous EtOH. Add the 20% EtOH
swelling solution directly to the bottle and cap tightly.
2. Allow the beads to swell overnight for approximately 12 hrs at 4°C with
gentle rocking or rotation.
3. The final slurry of the beads following rehydration will be approximately
20% ProteoMiner beads in 20% aqueous EtOH. However, it is
recommended to measure and adjust the slurry to 20% beads before
aliquoting into columns or plates in order to obtain the correct settled
bead volumes provided in the table below. Carefully transfer the entire
slurry to a sterile graduated container (for example a 15 ml screwcap
conical tube). Cover the container and allow the beads to settle for
approximately 30 min. After settling, adjust the volume of the swelling
solution by carefully removing or adding additional swelling solution so
that the ratio of settled bead volume to total slurry volume is 20% beads
(for example, 2 ml of settled beads in 10 ml total slurry volume).
4. Store rehydrated beads at 4°C in a sealed container until ready for use.
Step 2 – Aliquoting Beads
1. Before aliquoting ProteoMiner beads into columns or plates, you must first
calculate the appropriate amount of beads to use for your application. The
ratio of protein to beads is crucial for optimal performance. The dynamic
range of the protein concentration in the sample is reduced when the
high-abundance proteins saturate their ligands and the low-abundance
proteins bind to a sufficient number of ligands to allow for enrichment.
It is recommended to use a 10:1 ratio of sample volume (with protein
concentration of >50 mg/ml) to settled bead volume. The following chart
should help you determine what volume of beads you will need for your
application.
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