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

prepare solutions:

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)

prepare plate:

1. multi-dispense buffer:
   • row A (blank): 150µl buffer
   • row B (donor): 50µl buffer
   • row C (acceptor): 100µl buffer
2. multi-dispense donor (D):
   • row B: 100µl
   • row D: 100µl
3. multi-dispense acceptor (A):
   • row C: 50µl
   • row D: 50µl
4. mix plate by shaking 10 @ 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 10 @ 1200 r.p.m.
6. repeat measurement twice

Notes

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