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
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
Status
- We've received gBlock
Experiment
Transfection Plan:
Plate 1
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Plate 2
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Well 1HEK293 | Well 2HEK293 + Dummy (2500ng) | Well 3HEK293 + Dummy (1500ng) +hEF1a:YFP (1000ng) | Well 4 | Well 5 | Well 6 |
Well 7HEK293 +hEF1a:YFP (1000ng) +hEF1a:rtTA (500ng) +TRE:TEVp-Cofilin (1000ng) 0nM Dox | Well 8HEK293 +hEF1a:YFP (1000ng) +hEF1a:rtTA (500ng) +TRE:TEVp-Cofilin (1000ng) 1nM Dox | Well 9HEK293 +hEF1a:YFP (1000ng) +hEF1a:rtTA (500ng) +TRE:TEVp-Cofilin (1000ng) 10nM Dox | Well 10HEK293 +hEF1a:YFP (1000ng) +hEF1a:rtTA (500ng) +TRE:TEVp-Cofilin (1000ng) 100nM Dox | Well 11HEK293 +hEF1a:YFP (1000ng) +hEF1a:rtTA (500ng) +TRE:TEVp-Cofilin (1000ng) 1,000nM Dox | Well 12HEK293 +hEF1a:YFP (1000ng) +hEF1a:rtTA (500ng) +TRE:TEVp-Cofilin (1000ng) 2,000nM Dox |
Well 13 | Well 14 | Well 15 | Well 16 | Well 17 | Well 18 |
Well 19 | Well 20 | Well 21 | Well 22 | Well 23 | Well 2 |
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 680-RD | 1/15000 | https://licor.secure.force.com/catalog/LI_ProductDetailsPage?sku=926-68070&store=bio&viewState=DetailView | |
| Goat anti-rabbit | IRDye 800-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 + GFP + Dummy | HEK293 + GFP + TEVp-Cofilin |
| 50 | TEVp-Cofilin | |||
| 40 | GAPDH | GAPDH | GAPDH | GAPDH |
| 30 | YFP | |||
| 20 | Cofilin | Cofilin | Cofilin |
Status:
- Not yet started - waiting on parts