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
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
- dilute donor protein to 0.75µM in HBSP+
- dilute acceptor protein to 0.9µM in HBSP+
- dilute Rapamycin to 112.5µM
- adjust plate reader (set excitation to donor absorption, and emission to acceptor emission peak)
- multi-dispense buffer:
- row A (blank): 150µl buffer
- row B (donor): 50µl buffer
- row C (acceptor): 100µl buffer
- multi-dispense donor (D):
- row B: 100µl
- row D: 100µl
- multi-dispense acceptor (A):
- row C: 50µl
- row D: 50µl
- mix plate by shaking 10 @ 1200 r.p.m.
- load plate and measure acceptor emission after donor excitation for all wells
- remove plate
- multi-dispense 30µl rapamycin into a PCR stripe
- copy 2µl rapamycin into each well using a multi-channel pipette
- mix plate by shaking 10 @ 1200 r.p.m.
- repeat measurement twice
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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References
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