Description:
In this experiment, we want to want to compare the levels of endogenous cofilin and cofilin-eYFP 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 cofilin-eYFP 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 cofilin-eYFP in the cells.
Transfection Plan:
Well 1HEK293 | Well 2HEK293 + Dummy (1,000ng) | Well 3HEK293 + Dummy (500ng) +hEF1a:GFP (500ng) | Well 4 | Well 5 | Well 6 | ||
Well 7
| HEK293 +hEF1a:GFP (400ng) +hEF1a:rtTA (200ng) +TRE:Cofilin-TEVp (400ng) 0x Dox | Well 8HEK293 +hEF1a:GFP (500ng) +hEF1a:rtTA (200ng) +TRE:Cofilin-TEVp (500ng) 1x Dox | Well 9HEK293 +hEF1a:GFP (500ng) +hEF1a:rtTA (200ng) +TRE:Cofilin-TEVp (500ng) 10x Dox | Well 10HEK293 +hEF1a:GFP (500ng) +hEF1a:rtTA (200ng) +TRE:Cofilin-TEVp (500ng) 100x Dox | Well 11HEK293 +hEF1a:GFP (500ng) +hEF1a:rtTA (200ng) +TRE:Cofilin-TEVp (500ng) 1,000x Dox | Well 12HEK293 +hEF1a:GFP (500ng) +hEF1a:rtTA (200ng) +TRE:Cofilin-TEVp (500ng) 2,000x Dox | |
Well 13 | Well 14 | Well 15 | Well 16 | Well 17 | Well 18 | ||
Well 19 | Well 20 | Well 21 | Well 22 | Well 23 | Well 24 |
Stuff:
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 | Size (kDa) |
|---|---|
| Cofilin | 18.5 |
| Cofilin-TEVp | 48.6 |
| GAPDH | 37 |
| GFP | 27 |
Expected Results:
| Ladder | HEK293 | HEK293 + Dummy | HEK293 + GFP + Dummy | HEK293 + GFP + Cofilin-TEVp |
| 50 | Cofilin-TEVp | |||
| 40 | GAPDH | GAPDH | GAPDH | GAPDH |
| 30 | GFP | |||
| 20 | Cofilin | Cofilin | Cofilin |
http://www.thermoscientificbio.com/uploadedFiles/Resources/general-recommendations-for-sds-page.pdf