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

Introduction

Today is the big day!

Part 1: BRET Assay

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	GF Conc (μM)	Vol of BB (μL)	Tube label	GF Conc (μM)	Inhib Conc (μM)	Vol of BB + GF (μL)
DR1	100	150	DI1	100	10	150
DR2	10	135	DI2	100	1	135
DR3	1	135	DI3	100	0.1	135
DR4	0.1	135	DI4	100	0.01	135
DR5	0.01	135	DI5	100	0.001	135

3. Obtain your growth factor and inhibitor of interest from the front bench.
   • All growth factors are at a stock concentration of 10 mM.
   • All inhibitors are resuspended in DMSO at a concentration of 10 mM.
4. Prepare the dose-response tubes:
   • Add 1.5 μL of growth factor to tube DR1.
   • Mix by vortexing.
   • Remove 15 &mu:L from DR1 and put into DR2
   • Mix by vortexing.
   • Repeat until you get to DR5.
   • Do not add growth factor to DR6