Prbbbb:in vitro FRET FRB FKBP v1: Difference between revisions
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===Sensitized emission=== | ===Sensitized emission=== | ||
plate layout (5 or 6 replicas each): | '''plate layout (5 or 6 replicas each)''': | ||
* row A: 150µl blank | * row A: 150µl blank | ||
* row B (D): 50µl buffer + 100µl donor | * row B (D): 50µl buffer + 100µl donor | ||
Line 22: | Line 22: | ||
* row D (A+D): 100µl donor + 50µl acceptor | * row D (A+D): 100µl donor + 50µl acceptor | ||
prepare solutions: | '''prepare solutions''': | ||
# dilute donor protein to 0.75µM in HBSP+ | # dilute donor protein to 0.75µM in HBSP+ | ||
# dilute acceptor protein to 0.9µM in HBSP+ | # dilute acceptor protein to 0.9µM in HBSP+ | ||
Line 28: | Line 28: | ||
# adjust plate reader (set excitation to donor absorption, and emission to acceptor emission peak) | # adjust plate reader (set excitation to donor absorption, and emission to acceptor emission peak) | ||
prepare plate: | '''prepare plate''': | ||
# multi-dispense buffer: | # multi-dispense buffer: | ||
#* row A (blank): 150µl buffer | #* row A (blank): 150µl buffer | ||
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# mix plate by shaking 10'' @ 1200 r.p.m. | # mix plate by shaking 10'' @ 1200 r.p.m. | ||
measure w/o, with Rapamycin: | '''measure w/o, with Rapamycin''': | ||
# load plate and measure acceptor emission after donor excitation for all wells | # load plate and measure acceptor emission after donor excitation for all wells | ||
# remove plate | # remove plate |
Revision as of 07:45, 25 May 2010
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:
- 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)
prepare plate:
- 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.
measure w/o, with Rapamycin:
- 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!
raik:share your experience!
References
Contact
or instead, discuss this protocol.