20.109(F13): Mod 2 Day 7 HTS and Analysis

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(Part 1: Set-up BRET Assay Plate)
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==Introduction==
==Introduction==
Today is the big day!  
Today is the big day!  
 +
 +
Note why we use EDTA versus trypsin/EDTA for this protocol.
==Part 1: Set-up BRET Assay Plate==
==Part 1: Set-up BRET Assay Plate==
''Please read all the way through this protocol before starting your work!''
''Please read all the way through this protocol before starting your work!''
-
 
-
The cells that you transfected last time have been serum starved overnight to reduce any background SH2-EGFR interactions due to the presence of growth factors in serum. Similar to the cell stimulation done on M2D4 for our Western blot analysis, all steps will be completed in the main lab -- no sterile technique is required today.
 
-
 
#Two datasets will be obtained today:  
#Two datasets will be obtained today:  
Line 202: Line 201:
==Part 2: Prep cells for BRET Assay==
==Part 2: Prep cells for BRET Assay==
 +
The cells that you transfected last time have been serum starved overnight to reduce any background SH2-EGFR interactions due to the presence of growth factors in serum. Similar to the cell stimulation done on M2D4 for our Western blot analysis, all steps will be completed in the main lab -- no sterile technique is required today.
 +
 +
#Grab your 3, 60mm dishes of CHO-K1 cells transfected last time from the TC incubator and bring them to your lab bench.
 +
#You can find PBS and 0.5 mM EDTA on the front bench.
 +
#Aspirate the cell culture media from each plate.
 +
#Rinse your cells carefully with 3 mL of PBS -- remember to aim at the side of the plate and not directly towards the cells!
 +
#Remove the PBS and add 0.5 mL of 0.5 mM EDTA to each plate.
 +
#Bring the plates to the 37C incubator at the back of the lab (yes, the one that you used for bacterial culture) and incubate for 5 min.
 +
#During your 5 min incubation, prepare 3 - 15 mL conical tubes to spin down your cells and 3 - 1.5 mL tubes for cell counting.
 +
#After the 5 min incubation:
 +
#*Add 3.5mL of BRET buffer to each plate and resuspend the cells by triterating well.
 +
#*Add the total volume of 4mL cell suspension to the pre-labeled 15 mL conical tubes.
 +
#*Save 20 μL of cell suspension (put into the pre-labeled 1.5 mL tubes)
 +
#*Put your eppendorf tubes in the ice bucket at the front of the room.

Revision as of 22:21, 31 August 2013

20.109(F13): Laboratory Fundamentals of Biological Engineering

Home        Schedule Fall 2013        Assignments       
DNA Engineering        System Engineering        Biomaterials Engineering              

Introduction

Today is the big day!

Note why we use EDTA versus trypsin/EDTA for this protocol.

Part 1: Set-up BRET Assay Plate

Please read all the way through this protocol before starting your work!

  1. Two datasets will be obtained today:
    1. A dose-response (DR) curve of growth factor vs. SH2-EGFR interaction -- the growth factor concentration will vary (no drug).
    2. A dose-inhibition (DI) curve of inhibitor vs. SH2-EGFR interaction -- the drug concentration will vary (constant growth factor).
  2. Set-up 6 eppendorf tubes for each dataset as follows:
    • Label each tube and add the indicated volume of BRET Buffer (BB) to the tubes labeled DR1-6 only.
    • The final concentration of growth factor and inhibitor will be used later for your calculations.


Tube label Final GF Conc (μM) Vol of BB (μL) Tube label Final GF Conc (μM) Final Inhib Conc (μM) Vol of BB + GF (μL)
DR1 100 450 DI1 100 10 450
DR2 10 405 DI2 100 1 405
DR3 1 405 DI3 100 0.1 405
DR4 0.1 405 DI4 100 0.01 405
DR5 0.01 405 DI5 100 0.001 405
DR6 0 405 DI6 100 0 405
  1. Obtain your growth factor and inhibitor of interest from the front bench.
    • All growth factors are at a stock concentration of 30 mM.
    • All inhibitors are resuspended in DMSO at a concentration of 30 mM.
  2. Prepare the dose-response tubes by diluting 10x into each tube:
    • Add 4.5 μL of growth factor to tube DR1.
    • Mix by vortexing.
    • Remove 45 μL from DR1 and put into DR2
    • Mix by vortexing.
    • Repeat until you get to DR5.
    • Do not add growth factor to DR6
  3. Prepare the growth factor solution for the dose-inhibition dataset:
    • Add 30&mu:L of growth factor to 2.7 mL of BRET Buffer
    • Add the volume of BB + GF to each DI tube as indicated in the above chart
  4. Add the inhibitor to the dose-inhibitor tubes by diluting 10x into each tube:
    • Add 4.5 μL of inhibitor to tube DI1.
    • Mix by vortexing.
    • Remove 45 μL from DI1 and put into DI2
    • Mix by vortexing.
    • Repeat until you get to DI5.
    • Do not add inhibitor to DI6
    • Instead of inhibitor, add 4.5 &mu:L of DMSO to DI6 (find DMSO on the front bench)
  5. Pick up an opaque 96-well plate from the front bench. This will be your assay plate.
  6. Follow the plate map below to add growth factor and inhibitor to the appropriate wells:
1 2 3 4 5 6 7 8 9 10 11 12
A DR6 - SH2+EGFR DR6 - SH2+EGFR DR5 - SH2+EGFR DR5 - SH2+EGFR DR4 - SH2+EGFR DR4 - SH2+EGFR DR3 - SH2+EGFR DR3 - SH2+EGFR DR2 - SH2+EGFR DR2 - SH2+EGFR DR1 - SH2+EGFR DR1 - SH2+EGFR
B DR6 -
SH2 only
DR6 -
SH2 only
DR5 -
SH2 only
DR5 -
SH2 only
DR4 -
SH2 only
DR4 -
SH2 only
DR3 -
SH2 only
DR3 -
SH2 only
DR2 -
SH2 only
DR2 -
SH2 only
DR1 -
SH2 only
DR1 -
SH2 only
C DR6 - RLuc+EGFR DR6 - RLuc+EGFR DR5 - RLuc+EGFR DR5 - RLuc+EGFR DR4 - RLuc+EGFR DR4 - RLuc+EGFR DR3 - RLuc+EGFR DR3 - RLuc+EGFR DR2 - RLuc+EGFR DR2 - RLuc+EGFR DR1 - RLuc+EGFR DR1 - RLuc+EGFR
D DI6 - SH2+EGFR DI6 - SH2+EGFR DI5 - SH2+EGFR DI5 - SH2+EGFR DI4 - SH2+EGFR DI4 - SH2+EGFR DI3 - SH2+EGFR DI3 - SH2+EGFR DI2 - SH2+EGFR DI2 - SH2+EGFR DI1 - SH2+EGFR DI1 - SH2+EGFR
E DI6 -
SH2 only
DI6 -
SH2 only
DI5 -
SH2 only
DI5 -
SH2 only
DI4 -
SH2 only
DI4 -
SH2 only
DI3 -
SH2 only
DI3 -
SH2 only
DI2 -
SH2 only
DI2 -
SH2 only
DI1 -
SH2 only
DI1 -
SH2 only
F DI6 - RLuc+EGFR DI6 - RLuc+EGFR DI5 - RLuc+EGFR DI5 - RLuc+EGFR DI4 - RLuc+EGFR DI4 - RLuc+EGFR DI3 - RLuc+EGFR DI3 - RLuc+EGFR DI2 - RLuc+EGFR DI2 - RLuc+EGFR DI1 - RLuc+EGFR DI1 - RLuc+EGFR
  1. Once all wells have been filled with the appropriate contents, cover the plate with parafilm to prevent evaporation.

Part 2: Prep cells for BRET Assay

The cells that you transfected last time have been serum starved overnight to reduce any background SH2-EGFR interactions due to the presence of growth factors in serum. Similar to the cell stimulation done on M2D4 for our Western blot analysis, all steps will be completed in the main lab -- no sterile technique is required today.

  1. Grab your 3, 60mm dishes of CHO-K1 cells transfected last time from the TC incubator and bring them to your lab bench.
  2. You can find PBS and 0.5 mM EDTA on the front bench.
  3. Aspirate the cell culture media from each plate.
  4. Rinse your cells carefully with 3 mL of PBS -- remember to aim at the side of the plate and not directly towards the cells!
  5. Remove the PBS and add 0.5 mL of 0.5 mM EDTA to each plate.
  6. Bring the plates to the 37C incubator at the back of the lab (yes, the one that you used for bacterial culture) and incubate for 5 min.
  7. During your 5 min incubation, prepare 3 - 15 mL conical tubes to spin down your cells and 3 - 1.5 mL tubes for cell counting.
  8. After the 5 min incubation:
    • Add 3.5mL of BRET buffer to each plate and resuspend the cells by triterating well.
    • Add the total volume of 4mL cell suspension to the pre-labeled 15 mL conical tubes.
    • Save 20 μL of cell suspension (put into the pre-labeled 1.5 mL tubes)
    • Put your eppendorf tubes in the ice bucket at the front of the room.
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