Prbbbb:in vitro FRET FRB FKBP v1

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# mix plate by shaking 10'' @ 1200 r.p.m.
# mix plate by shaking 10'' @ 1200 r.p.m.
# repeat measurement twice
# repeat measurement twice
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==Notes==
==Notes==

Revision as of 10:41, 25 May 2010

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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

plate layout (5 or 6 replicas each):

  • row A: 150µl blank
  • row B (D): 50µl buffer + 100µl donor
  • row C (A): 100µl buffer + 50µl acceptor
  • row D (A+D): 100µl donor + 50µl acceptor
  1. dilute donor protein to 0.75µM in HBSP+
  2. dilute acceptor protein to 0.9µM in HBSP+
  3. dilute Rapamycin to 112.5µM
  4. adjust plate reader (set excitation to donor absorption, and emission to acceptor emission peak)
  5. multi-dispense buffer:
    • row A (blank): 150µl buffer
    • row B (donor): 50µl buffer
    • row C (acceptor): 100µl buffer
  1. multi-dispense donor (D):
    • row B: 100µl
    • row D: 100µl
  1. multi-dispense acceptor (A):
    • row C: 50µl
    • row D: 50µl
  1. mix plate by shaking 10 @ 1200 r.p.m.
  2. load plate and measure acceptor emission after donor excitation for all wells
  3. remove plate
  4. multi-dispense 30µl rapamycin into a PCR stripe
  5. copy 2µl rapamycin into each well using a multi-channel pipette
  6. mix plate by shaking 10 @ 1200 r.p.m.
  7. repeat measurement twice

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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