Group C: Levels of Endogenous Cofilin

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.

Transfection Plan:

Stuff:

Controls:

GAPDH: Loading control (how many cells we've loaded onto the gel)

GFP: Transfection marker (how many transfected cells we've loaded onto the gel)

Protein Sizes:

Gels:

We have the any kD gels in the lab (expire 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.

Expected Results:

http://www.thermoscientificbio.com/uploadedFiles/Resources/general-recommendations-for-sds-page.pdf