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

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(New page: {{Template:20.109(F13)}} <div style="padding: 10px; width: 640px; border: 5px solid #66399F;"> ==Introduction== Today is the big day! ==Part 1: BRET Assay== ''Please read all the way thr...)
 
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''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 for you to reduce any background SH2-EGFR interactions due to the presence of 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.
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.


#Obtain your growth factor and inhibitor of interest from the front bench.
#Obtain your growth factor and inhibitor of interest from the front bench.
#*All growth factors are at a stock concentration of 100 &mu;g/mL.
#*All growth factors are at a stock concentration of 100 mM.
#*All inhibitors are resuspended in DMSO at a concentration of 10 &mu;M.
#*All inhibitors are resuspended in DMSO at a concentration of 10 mM.
#Two datasets will be obtained today:  
#Two datasets will be obtained today:  
##A dose-response (DR) curve of growth factor vs. SH2-EGFR interaction -- the growth factor concentration will vary (no drug).
##A dose-response (DR) curve of growth factor vs. SH2-EGFR interaction -- the growth factor concentration will vary (no drug).
##A dose-inhibition (DI) curve of inhibitor vs. SH2-EGFR interaction -- the drug concentration will vary (constant growth factor).
##A dose-inhibition (DI) curve of inhibitor vs. SH2-EGFR interaction -- the drug concentration will vary (constant growth factor).
#Label two sets of 6 eppendorf tubes:
#Set-up 6 eppendorf tubes for each dataset as follows:
#*DR 1-6
#*Label each tube and add the appropriate amount of BRET Buffer (BB).
#*DI 1-6
#*The final concentration of growth factor and inhibitor will be used later for your calculations.
#
<center>
{| border="1"
! Tube label
! GF Conc (&mu;M)
! Vol of BB (&mu;L)
! Tube label
! GF Conc (&mu;M)
! Inhib Conc (&mu;M)
! Vol of BB (&mu;L)
|-
|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
|-}
</center>

Revision as of 13:42, 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!

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. Obtain your growth factor and inhibitor of interest from the front bench.
    • All growth factors are at a stock concentration of 100 mM.
    • All inhibitors are resuspended in DMSO at a concentration of 10 mM.
  2. 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).
  3. Set-up 6 eppendorf tubes for each dataset as follows:
    • Label each tube and add the appropriate amount of BRET Buffer (BB).
    • 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 (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
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