Prbbbb:in vitro FRET FRB FKBP v1

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(New page: Back to all protocols ==Overview== This protocol describes how to measure FRET efficiencies for a chemically induced interaction between FRB and FKBP12. A first mea...)
Current revision (03:17, 24 August 2011) (view source)
(Procedure)
 
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==Procedure==
==Procedure==
-
'''Sensitized emission'''
+
===Sensitized emission===
-
# dilute donor protein to 0.75µM in HBSP+
+
''Final conditions: 0.5µM donor + 0.3µM acceptor + 1.5µM Rapamycin in HBSP+''
-
# dilute acceptor protein to 0.9µM 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)
 +
# dilute donor protein to 0.75µM in HBSP+; V=1400µl (1200+reserve)
 +
# dilute acceptor protein to 0.9µM in HBSP+; V=720µl (600+reserve)
 +
# dilute Rapamycin to 112.5µM; V=100µl (50+reserve)
 +
# 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 10s @ 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 10s @ 1200 r.p.m.
 +
# repeat measurement twice
 +
 
 +
===Donor quenching===
 +
 
 +
''Final conditions: 0.3µM donor + 0.5µM acceptor + 1.5µM 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''':
 +
# dilute donor protein to 0.45µM in HBSP+
 +
# dilute acceptor protein to 1.5µM in HBSP+
# dilute Rapamycin to 112.5µM
# dilute Rapamycin to 112.5µM
 +
# 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===
-
# add 500 µl buffer I to the pellet
+
to be described.
-
# resuspend by pipetting or 2 times 5-10 s vortexing
+
-
# boil and load 5 µl + loading buffer on a SDS PAGE gel
+
-
# continue with purification using the same buffer without Triton and with varying amounts of Imidazole
+
==Notes==
==Notes==

Current revision

Back to all protocols

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

Final conditions: 0.5µM donor + 0.3µM acceptor + 1.5µM 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:
    • 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 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 + 1.5µM 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.

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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