Chemistry & Biology
Bead to Microarray Screening
reached 2500 rpm, or by pressing ‘‘start’’ and then ‘‘stop’’ as soon as the
speed reached 2500 rpm.
cence values for each of the spots were cut and pasted in Excel and arranged
(using simple macros) to simplify transferring results to GraphPad Prism 5.0
software for binding curve analyses.
While visualizing the clump of hit beads on the bottom of the tube, most of
the dynabeads and TBST was drained from the top using a 1000 ml pipette-
man. HPLC water (1 ml) was added to the tube, and the tube inverted and
spun down as before. Again, most of the solution was drained while taking
great care not to suck up the beads from the bottom of the tube. This washing
step was repeated six times. For hit beads from libraries containing the methi-
onine linker, most of the water was drained, and 1 ml acetonitrile was added.
Beads were then transferred to a 96 well plate and sorted one bead per well
under a dissecting microscope. This can be quite tedious or simple, depending
on technique (>100 hits can be sorted in less than 1 hr by the technique
described in the Supplemental Information). At this point, 20 ml of 30 mg/ml
CNBr in 5:4:1 acetonitrile:AcOH:water was added per well, and the plate
covered with sticky foil and placed on a shaker at RT overnight. The next
day, the foil was removed and the 96 well plate left to air dry in a chemical
hood for several hours. HPLC-grade water (20 ml) was added, and the plate
covered and left on a shaker for 1 hr at RT; 10 ml from each well was transferred
to a 384 well plate containing 10 ml/well DMSO, and the plate sealed and set
aside for microarray spotting. Acetonitrile (10 ml) was added to each of the
wells in the 96 well plate containing hit beads. This plate was sealed and set
aside for MS sequencing. For hit beads containing the Asp-Pro linker, after
the water washing of hit beads to remove most of the Dynabeads and TBST,
beads were resuspended in HPLC-grade water and transferred to a small Petri
dish under a dissecting microscope. A 10 ml pipetteman was set at 1 ml, and
beads were transferred one bead at a time to thin-walled PCR tubes; 20 ml
SUPPLEMENTAL INFORMATION
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health Director’s Pioneer
Award (DP1OD000663).
Received: October 12, 2009
Revised: December 8, 2009
Accepted: December 8, 2009
Published: January 28, 2010
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1
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44 Chemistry & Biology 17, 38–45, January 29, 2010 ª2010 Elsevier Ltd All rights reserved
Astle, John M.
