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.
Results:
Expand | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Expand | ||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||||||||||||||||||||||||||||||
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
Next step: I would like to have a positive control. Abcam suggests HeLa lysates (if we have those around in the lab) |