Thoughts on this experimental design - CAT elisa (Jun/16/2005 )
Just wondering what people thought of this experimental design for a CAT elisa assay.
I've got CAT plasmids that contain a wild type stem loop and poly T tract, a plasmid with both SL and polyT removed, with just the SL removed, and just the poly T removed. Also have plasmids with mutations in the SL and poly T. I've got beta actin CAT plasmid, and a beta actin Luciferase plasmid.
Now, i want to see how estrogen will effect the expression of these stem loopy plasmids. So, in MCF-7 cells, the stem loopy plasmids (all CAT) and beta actin Luciferase plasmids will be cotransfected. They'll then be exposed to estrogen for various amounts of time. The positive control would be the wild type stem loop, but a negative control? (HELP!!!) would that be the beta actin CAT ? (please say yes)
I then take a reading of the luciferase, and the run an elisa for the CAT. Normalise the results, repeat X2 , and viola, done.
Someone in my building was talking about using empty vectors, and i lost it after that. do i need an empty vector of luciferase and an empty vector of CAT, or just of the CAT... don't get this part at all. Anythoughts (mine are all jumbled at the moment) would be really useful.
beta actin-CAT can act as a positive control in any CAT based expt (sorry).
in your case, yes, the empty vector(without minimal promoter too, with it you will get little expression) i.e. promoterless CAT will act as negative control. the second (not negative exactly but will do) control would be transfected contruct without estrogen treatment. if estrogen affects the CAT expression, you can plot % activation in presence or absence of estrogen for various constructs.