Prbbbb:in vitro FRET FRB FKBP v1

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Contents

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

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

raik:share your experience!


References

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