Bio-Rad Bio-Beads SM-2 Adsorbents - Manual (Page 7)

2. Incubate the beads with the solution at room
temperature on a rotary platform to insure thorough
mixing. Stir for 30 minutes.
3. Centrifuge the mixture, and pipette out the
supernatent.
4. Free fluorescent dye will reappear upon storage, so
samples should be adsorbed with the Bio-Beads SM-
2 adsorbent an hour before use in immunofluorescent
staining experiments.
4.4 Column Protocol
1. Slurry 2 g of Bio-Beads SM-2 adsorbent in
phosphate buffer.
2. Pour a 0.8 x 4 cm Poly-Prep ® column containing a
1 ml bed volume of Bio-Beads adsorbent.
3. Apply 1–2 ml solution containing the fluorescent-
labeled antibody, and collect the effluent.
4. Wash the column with PBS and collect the effluent.
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3. Wash with 10 mM phosphate buffer at 0.03 ml/min.
Collect fractions and assay for protein.
The column can be regenerated by washing in 4 bed
volumes of methanol, followed by rinsing in DI water.
4.2 Adsorption of Unconjugated
Fluorescent Dye
This procedure was used by Spack et al. to reduce
high background fluorescence with rhodamine-labeled
antibodies.
24 It should be useful for many fluorescent
labels. The Bio-Beads SM-2 adsorbent was prewashed
with 2–3 bed volumes of methanol, distilled water, and
PBS, and stored in PBS until use. If trapped air within
the pores of the Bio-Beads adsorbent causes the
adsorbent to float, the slurried beads may be degassed to
correct this.
4.3 Batch Protocol
1. Add 0.1 g of Bio-Beads adsorbent to 1–2 ml of
solution containing the fluorescent-labeled antibody.
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