Prbbbb:in vitro FRET FRB FKBP v1

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(Procedure)
Current revision (03:17, 24 August 2011) (view source)
(Procedure)
 
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==Procedure==
==Procedure==
-
'''Sensitized emission'''
+
===Sensitized emission===
-
plate layout (5 or 6 replicas each):
+
''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 A: 150µl blank
* row B (D): 50µl buffer + 100µl donor
* row B (D): 50µl buffer + 100µl donor
Line 22: Line 24:
* row D (A+D): 100µl donor + 50µl acceptor
* row D (A+D): 100µl donor + 50µl acceptor
-
# dilute donor protein to 0.75µM in HBSP+
+
'''prepare solutions''':
-
# dilute acceptor protein to 0.9µM in HBSP+
+
(volumes are for one set of 6 replicas)
-
# dilute Rapamycin to 112.5µM
+
# 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)
# adjust plate reader (set excitation to donor absorption, and emission to acceptor emission peak)
 +
 +
'''prepare plate''':
# multi-dispense buffer:
# multi-dispense buffer:
-
** row A (blank): 150µl buffer
+
#* row A (blank): 150µl buffer
-
** row B (donor): 50µl buffer
+
#* row B (donor): 50µl buffer
-
** row C (acceptor): 100µl buffer
+
#* row C (acceptor): 100µl buffer
# multi-dispense donor (D):
# multi-dispense donor (D):
-
** row B: 100µl
+
#* row B: 100µl
-
** row D: 100µl
+
#* row D: 100µl
# multi-dispense acceptor (A):
# multi-dispense acceptor (A):
-
** row C: 50µl
+
#* row C: 50µl
-
** row D: 50µl
+
#* row D: 50µl
-
# mix plate by shaking 10'' @ 1200 r.p.m.
+
# 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
# load plate and measure acceptor emission after donor excitation for all wells
# remove plate
# remove plate
# multi-dispense 30µl rapamycin into a PCR stripe
# multi-dispense 30µl rapamycin into a PCR stripe
# copy 2µl rapamycin into each well using a multi-channel pipette
# copy 2µl rapamycin into each well using a multi-channel pipette
-
# mix plate by shaking 10'' @ 1200 r.p.m.
+
# mix plate by shaking 10s @ 1200 r.p.m.
# repeat measurement twice
# 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
 +
# 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==
==Notes==

Current revision

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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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