20.109(F13): Mod 2 Day 7 HTS and Analysis: Difference between revisions
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{| border = "1" class="wikitable" style="margin: 1em auto 1em auto;" | {| border = "1" class="wikitable" style="margin: 1em auto 1em auto;" | ||
! Tube label | ! Tube label | ||
! GF Conc (μM) | ! Final GF Conc (μM) | ||
! Vol of BB (μL) | ! Vol of BB (μL) | ||
! Tube label | ! Tube label | ||
! GF Conc (μM) | ! Final GF Conc (μM) | ||
! Inhib Conc (μM) | ! Final Inhib Conc (μM) | ||
! Vol of BB + GF (μL) | ! Vol of BB + GF (μL) | ||
|- | |- | ||
|DR1 | |DR1 | ||
|100 | |100 | ||
| | |450 | ||
|DI1 | |DI1 | ||
|100 | |100 | ||
|10 | |10 | ||
| | |450 | ||
|- | |- | ||
|DR2 | |DR2 | ||
|10 | |10 | ||
| | |405 | ||
|DI2 | |DI2 | ||
|100 | |100 | ||
|1 | |1 | ||
| | |405 | ||
|- | |- | ||
|DR3 | |DR3 | ||
|1 | |1 | ||
| | |405 | ||
|DI3 | |DI3 | ||
|100 | |100 | ||
|0.1 | |0.1 | ||
| | |405 | ||
|- | |- | ||
|DR4 | |DR4 | ||
|0.1 | |0.1 | ||
| | |405 | ||
|DI4 | |DI4 | ||
|100 | |100 | ||
|0.01 | |0.01 | ||
| | |405 | ||
|- | |- | ||
|DR5 | |DR5 | ||
|0.01 | |0.01 | ||
| | |405 | ||
|DI5 | |DI5 | ||
|100 | |100 | ||
|0.001 | |0.001 | ||
| | |405 | ||
|- | |||
|DR6 | |||
|0 | |||
|405 | |||
|DI6 | |||
|100 | |||
|0 | |||
|405 | |||
|- | |- | ||
|} | |} | ||
#<li value = "3">Obtain your growth factor and inhibitor of interest from the front bench. | #<li value = "3">Obtain your growth factor and inhibitor of interest from the front bench. | ||
#*All growth factors are at a stock concentration of | #*All growth factors are at a stock concentration of 30 mM. | ||
#*All inhibitors are resuspended in DMSO at a concentration of | #*All inhibitors are resuspended in DMSO at a concentration of 30 mM. | ||
#Prepare the dose-response tubes: | #Prepare the dose-response tubes by diluting 10x into each tube: | ||
#*Add | #*Add 4.5 μL of growth factor to tube DR1. | ||
#*Mix by vortexing. | #*Mix by vortexing. | ||
#*Remove | #*Remove 45 μL from DR1 and put into DR2 | ||
#*Mix by vortexing. | #*Mix by vortexing. | ||
#*Repeat until you get to DR5. | #*Repeat until you get to DR5. | ||
#*<font color = red>'''Do not add growth factor to DR6'''</font color> | #*<font color = red>'''Do not add growth factor to DR6'''</font color> | ||
#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 | |||
#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. | |||
#*<font color = red>'''Do not add inhibitor to DI6'''</font color> | |||
#*Instead of inhibitor, add 4.5 &mu:L of DMSO to DI6 (find DMSO on the front bench) | |||
#Pick up an opaque 96-well plate from the front bench. This will be your assay plate. | |||
#Follow the plate map below to add growth factor and inhibitor to the appropriate wells. | |||
Revision as of 18:08, 31 August 2013
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.
- 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-inhibition (DI) curve of inhibitor vs. SH2-EGFR interaction -- the drug concentration will vary (constant growth factor).
- 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 |
- 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.
- 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
- 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
- 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)
- Pick up an opaque 96-well plate from the front bench. This will be your assay plate.
- Follow the plate map below to add growth factor and inhibitor to the appropriate wells.
