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 | - | T1:1/10000 T2:1/2500 | 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 | YFP | ||
| 20 | Cofilin | Cofilin | Cofilin | Cofilin |
Results:
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Odyssey blocked blot. Cofilin and TEVp-Cofilin bands are not there... We know that cofilin is present in relatively large amounts and the fact that neither native cofilin nor TEVp-cofilin bands appear might indicate that there is a problem with the anitbody or the antibody treatment of the blot. Both anti-cofilin and anti-eYFP are rabbit antibodies and since eYFP bands show, we can assume that the goat anti-rabbit secondary works. YFP and GAPDH bands are higher than they should be. Low signal from GAPDH (relative to YFP) | BSA blocked blot. Cofilin and TEVp-Cofilin are not here either There's a lot of background | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Modifications to Trial 1:
Increased cofilin primary antibody concentration from 1/10,000 to 1/2500 because both cofilin and TEVp-cofilin bands didn't show up in Trial 1
--- Didn't help that much considering the cofilin bands are still not there
Didn't probe for GAPDH because the amount of background that the 800 secondary antibody was producing
--- No background 
(although, there wasn't that much background with the odyssey blocked blot in Trial 1)
Also the YFP band (the only one there) is again slightly higher than it should be
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Next step: I would like to have a positive control. Abcam suggests HeLa lysates (if we have those around in the lab)