Description:
In this experiment, we want to want to compare the levels of endogenous cofilin and TEVp-Cofilin expressed under an inducible promoter with different levels of dox induction. This will allow us to determine what level of expression of cofilin would lead to the best signal:noise ratio of exogenous cofilin to endogenous cofilin. We are planning to transfect HEK293 with TEVp-Cofilin under a TRE promoter and induce with varying levels of dox. We will then preform a Western blot with an anti-cofilin primary antibody and compare band sizes to test for the relative amounts of endogenous cofilin and exogenous TEVp-Cofilin in the cells.
Cloning
COMPLETE
Description
Making pEXPR TRE:TEVp-cofilin.
Plan
- Order TEVp-cofilin
- Golden Gate gBlocks into ggDONR
- Transform bacteria with Golden Gate product and plate
- Pick colonies and grow in liquid culture
- Miniprep cells and verify product
- Make glycerol stocks
- LR into pDEST
- Transform, pick colonies and grow in liquid culture
- Miniprep cells and verify product
- Make glycerol stocks
- Midiprep product
Experiment
Transfection Plan:
Plate 1
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Plate 2
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Controls:
GAPDH: Loading control (how many cells we've loaded onto the gel)
YFP: Transfection marker (how many transfected cells we've loaded onto the gel)
Protein Sizes:
Protein | Size (kDa) |
|---|---|
| Cofilin | 18.5 |
| TEVp-Cofilin | 48.6 |
| GAPDH | 37 |
| YFP | 27 |
Gels:
We have the any kD gels in the lab (expires 2015) and this will work for both our western blot and antibody group's western blot.
If we run out of the above gel we are considering getting the 4-25% gel in order to optimize our resolution. This will also work for both our western blot and antibody group's western blot.
Antibodies:
Antibody | Type | Conjugation | Dilution Used | Link |
|---|---|---|---|---|
| Anti-Cofilin/Cofilin-TEVp | Rabbit Monoclonal | - | 1/10000 | http://www.abcam.com/cofilin-antibody-ep6376-ab134963.html |
| Anti-GAPDH | Mouse Monoclonal | - | 1/10000 | http://www.abcam.com/gapdh-antibody-mabcam-9484-loading-control-hrp-ab9482.html |
| Anti-eYFP | Rabbit Polyclonal | - | 1/10000 | http://www.abcam.com/gfp-antibody-chip-grade-ab290.html |
| Goat anti-mouse secondary | IRDye 800-RD | 1/15000 | https://licor.secure.force.com/catalog/LI_ProductDetailsPage?sku=926-68070&store=bio&viewState=DetailView | |
| Goat anti-rabbit | IRDye 680-CW | 1/15000 | https://licor.secure.force.com/catalog/LI_ProductDetailsPage?sku=827-08365&store=bio&viewState=DetailView |
Expected Results:
| Ladder | HEK293 | HEK293 + Dummy | HEK293 + YFP + Dummy | HEK293 + YFP + TEVp-Cofilin |
| 50 | TEVp-Cofilin | |||
| 40 | GAPDH | GAPDH | GAPDH | GAPDH |
| 30 | YFP | |||
| 20 | Cofilin | Cofilin | Cofilin | Cofilin |
Results: