Bio-Rad ProteoMiner Protein Enrichment Kits - Manual (Page 18)

Volume of sample Volume of 20% bead Resulting settled bead
available slurry required volume (after swelling
solution is removed)
200 µl 100 µl 20 µl
500 µl 250 µl 50 µl
1,000 µl 500 µl 100 µl
This chart assumes a protein concentration of >50 mg/ml, which is typical for
serum. If your sample concentration is lower, adjust volumes accordingly to
assure 0.5 mg of protein per µl of beads. It is not recommended to use less
than 10 mg of protein.
2. After determining the appropriate amount of beads to use for your
application, carefully pipette the bead slurry into either a spin column
(Bio-Rad catalog #732-6207) or 96-well plate with low-protein binding
membrane (Available from Pall Corp., part number 5039). It is
important to assure that the slurry is well mixed before
pipetting to assure reproducibility of aliquoting. If beads have
settled, lightly mix until resuspended.
Step 3 – Prepare Reagents
1. Prepare PBS wash buffer (150 mM NaCl, 10 mM NaH
2
PO
4
, pH 7.4).
Approximately 5 ml per sample will be required.
2. Prepare elution reagent(s). See pages 1 and 2 for elution reagent options.
Two elution protocols are available depending on your downstream
application. It is recommended that the majority of users follow the single
elution protocol that utilizes urea CHAPS/acetic acid (page 1). For SELDI
users and others who wish to sequentially elute their proteins, we
recommend the sequential elution protocol that utilizes four elution
reagents (see page 2). The sequential elution reagents are also available
for purchase (catalog #163-3003).
Step 4 – Sample Processing
1. Depending on which settled bead volume and which set of elution
reagents you have chosen, please refer to the appropriate protocol: single
elution protocol, 100 µl settled bead volume (Section 6); single elution
protocol, 20 µl settled bead volume (Section 7); sequential elution
protocol, 100 µl settled bead volume (Section 8); sequential elution
protocol, 20 µl settled bead volume (Section 9). These protocols are
designed for use with 100 µl or 20 µl of settled beads. If using different
amounts adjust accordingly. Additionally, if you choose to use a 96-well
filter plate, vacuum will have to be used in place of centrifugation.
2. After eluting your samples from the column/plate, refer to Section 11 for
information on preparing your sample for analysis.
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