Protocol

Binding assays were set up in 96-well polypropylene plates. All compounds were first diluted to 40 times final concentration in 100% DMSO and then threefold serial dilutions were made in 100% DMSO. Full-length Hsp90 isoforms were diluted to two times their final concentration in PBS

B

120 100 80 60 40 20 0

HSP90a

120 100 80 60 40 20 0

120

100

n o

80

-Q

60

In

40

20

0

HSP90ß

HSP90ß

120100806040200-

17AAG 13

17AAG 13

GRP94

120100806040200-

HSP90a

GRP94

FIGURE 5.3 (A) AlphaScreen™ Hsp90 isoform (full length Hsp90a, Hsp90j3, and Grp94) binding assay. (B) Linearity of Hsp90 isoform binding assay response. Hsp90 isoforms were diluted in two-fold increments and binding assays set up as described in Section 5.4. Hsp90a and Hsp90^ received 10 nM of b-GA and Grp94 received 30 nM of b-GA. All points represent an average of N = 2. (C) Determination of binding potency of GA and 17AAG for Hsp90a, Hsp90^, and Grp94 by AlphaScreen binding assays. Final assay conditions were 0.25 nM Hsp90a, 1 nM Hsp90j3, and 3 nM Grp94, combined with 10 nM biotin-GA for Hsp90a and Hsp90j3, and 30 nM biotin-GA for Grp94. Assays were read 4.5 hr post bead additions.

containing 0.1% BSA and 1 mM DTT, and 50 pL per well dispensed into plates, followed by 2.5 pL of DMSO or compounds. Biotin-GA was diluted to 2 times the final concentration and 50 pL per well added to the Hsp90 isoforms. The mixture was incubated for 2 to 2.5 hr at room temperature before 30 pL were transferred to 96-well, half-area white plates. AlphaScreen beads from the his-tidine detection kit were diluted to 40 pg/mL in PBS plus 0.1% BSA, and 30 pL were then added to the 30 pL in the half-area white plates. Plates were sealed and incubated at room temperature for 1 to 20 hr before reading in the Perkin Elmer Fusion-a instrument. AlphaScreen beads and plates containing beads were continuously kept under darkened conditions. A background signal was determined using a high concentration (10 pM final) of radicicol.

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