S. Datta et al. / Bioorg. Med. Chem. 18 (2010) 6099–6108
6107
expressed with 100
l
M IPTG at 16 °C over 16 h, whereas Hdmx
using the standard single-site competitive inhibition equation
(percent inhibition = 100[I]/([I] + IC50), where [I] is the concentra-
tion of the peptide inhibitor) in KaleidaGraph V.4 software (Syn-
ergy Software).
was overexpressed with 1 mM IPTG at 30 °C over 5 h. Upon centri-
fugation, the harvested cells were lysed in an extraction buffer (1ꢁ
PBS, 1 mM PMSF, and 10 mM imidazole, pH 7.9), supplemented
with 1ꢁ BugBuster™, and the lysate was cleared by centrifugation
at 12,000g for 30 min at 4 °C. The lysates were incubated with TA-
LON metal affinity resin (BD Biosciences) for 1 h at 4 °C, washed
with the extraction buffer, and treated with 300 mM imidazole in
1ꢁ PBS (pH 7.9). Purity of the eluants was assessed by SDS–PAGE,
their concentrations were measured by Bradford’s assay (Pierce
Biotechnology), and the aliquots were stored in 50% glycerol at
ꢂ20 °C.
Acknowledgments
This research was supported by the National Institutes of Heal-
th (1R21AI077482-01) and Walther Cancer Institute FDN, Inc.
M.E.B. was funded by Purdue Research Foundation grant.
Supplementary data
4.2.8. Affinity capture–elution assay
SICLOPPS fusions were expressed in the corresponding selection
strains in LB supplemented with 13 lM L-(+)-arabinose and chlor-
Supplementary data associated with this article can be found, in
amphenicol at 16 °C over 16 h. The induced cells were harvested
and lysed with an extraction buffer (1ꢁ PBS and 1 mM PMSF, pH
7.2) containing 1ꢁ BugBuster™. The lysate was incubated with chi-
tin beads (New England BioLabs, Inc.) at 4 °C for 1 h. The beads
were washed with 1ꢁ PBS, blocked with 0.1% bovine serum albu-
min (BSA) in 1ꢁ PBS, and incubated for 1.5 h with an equimolar
mixture of Hdm2 and Hdmx (1 mM each in 1ꢁ PBS and 1 mM
DTT, pH 7.2), followed by three PBST (1ꢁ PBS and 0.1% Tween
20, pH 7.2) washes. Peptide ETFSDLWKLL (1 mM in 1ꢁ PBS and
1 mM DTT, pH 7.2) was incubated with the washed beads for
16 h at room temperature, and the eluants along with the post-elu-
tion beads were analyzed by SDS–PAGE.
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