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

Introduction

Today is the big day!

Part 1: Set-up BRET Assay Plate

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.

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

3. 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.
4. 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
5. 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
6. 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)
7. Pick up an opaque 96-well plate from the front bench. This will be your assay plate.
8. 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