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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: 

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title Trial 1
Lane 1 2 3 4 5 6 7 8 9 10 11 Contents Ladder Untransfected Dummy eYFP eYFP rtTa TEVp-Cofilin 0nM Dox eYFP rtTa TEVp-Cofilin 1nM Dox eYFP rtTa TEVp-Cofilin 10nM Dox eYFP rtTa TEVp-Cofilin 100nM Dox eYFP rtTa TEVp-Cofilin 1000nM Dox eYFP rtTa TEVp-Cofilin 2000nM Dox Ladder Protein Size (kDa) Cofilin 18.5 TEVp-Cofilin  48.6 GAPDH 37 YFP 27                                                         Image Added Image Added Image Added 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

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title Trial 2
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 Lane 1 2 3 4 5 6 7 8 9 10 11 Contents Ladder Untransfected Dummy eYFP eYFP rtTa TEVp-Cofilin 0nM Dox eYFP rtTa TEVp-Cofilin 1nM Dox eYFP rtTa TEVp-Cofilin 10nM Dox eYFP rtTa TEVp-Cofilin 100nM Dox eYFP rtTa TEVp-Cofilin 1000nM Dox eYFP rtTa TEVp-Cofilin 2000nM Dox Ladder Protein Size (kDa) Cofilin 18.5 TEVp-Cofilin  48.6 GAPDH 37 YFP 27 Image Added Image Added Next step: I would like to have a positive control. Abcam suggests HeLa lysates (if we have those around in the lab)