Prbbbb:in vitro FRET FRB FKBP v1

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Overview

This protocol describes how to measure FRET efficiencies for a chemically induced interaction between FRB and FKBP12. A first measurement is done without rapamycin. Then rapamycin is added in three-fold excess. Sensitized (acceptor) emission is measured with an excess of donor (0.3µM acceptor + 0.5µM donor). Donor quenching is measured with an excess of acceptor (0.5µM donor + 0.3µM acceptor).

Materials

• 1x HBS-P+ with 50 µM EDTA -- our standard measurement buffer
• fluorescence plate reader
• black 96-well flat-bottom plates
• multi-dispensing pipette

Procedure

Sensitized emission

1. dilute donor protein to 0.75µM in HBSP+
2. dilute acceptor protein to 0.9µM in HBSP+
3. dilute Rapamycin to 112.5µM

1. add 500 µl buffer I to the pellet
2. resuspend by pipetting or 2 times 5-10 s vortexing
3. boil and load 5 µl + loading buffer on a SDS PAGE gel
4. continue with purification using the same buffer without Triton and with varying amounts of Imidazole

Notes

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