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

Sensitized emission
Final conditions: 0.5µM donor + 0.3µM acceptor + 2µM Rapamycin in HBSP+

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

prepare solutions: (volumes are for one set of 6 replicas)
 * 1) dilute donor protein to 0.75µM in HBSP+; V=1400µl (1200+reserve)
 * 2) dilute acceptor protein to 0.9µM in HBSP+; V=720µl (600+reserve)
 * 3) dilute Rapamycin to 112.5µM; V=100µl (50+reserve)
 * 4) adjust plate reader (set excitation to donor absorption, and emission to acceptor emission peak)

prepare plate:
 * 1) multi-dispense buffer:
 * 2) * row A (blank): 150µl buffer
 * 3) * row B (donor): 50µl buffer
 * 4) * row C (acceptor): 100µl buffer
 * 5) multi-dispense donor (D):
 * 6) * row B: 100µl
 * 7) * row D: 100µl
 * 8) multi-dispense acceptor (A):
 * 9) * row C: 50µl
 * 10) * row D: 50µl
 * 11) mix plate by shaking 10s @ 1200 r.p.m.

measure w/o, with Rapamycin:
 * 1) load plate and measure acceptor emission after donor excitation for all wells
 * 2) remove plate
 * 3) multi-dispense 30µl rapamycin into a PCR stripe
 * 4) copy 2µl rapamycin into each well using a multi-channel pipette
 * 5) mix plate by shaking 10s @ 1200 r.p.m.
 * 6) repeat measurement twice

Donor quenching
Final conditions: 0.3µM donor + 0.5µM acceptor + 2µM Rapamycin in HBSP+

plate layout -- same as above (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

prepare solutions:
 * 1) dilute donor protein to 0.45µM in HBSP+
 * 2) dilute acceptor protein to 1.5µM in HBSP+
 * 3) dilute Rapamycin to 112.5µM
 * 4) adjust plate reader (set excitation to donor absorption, and emission to donor emission peak)

Now follow the remaining steps of the sensitized acceptor measurements.

Analysis
to be described.

Contact

 * Raik

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