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[quipu]

Hi!

I'm trying to optimize my reporter assay with 3 vectors, expressionvector with transcription factor, reporter vector with luciferase and betagal vector.

I found that the luciferase activity (counts per second) is much higher whan betagal is also transfected, compared to just the other two vectors. I'm confused..... does betagal activates my reporter, or what?

the ratio between vehicle treated transfected cells and ligand treated transfected cells is almost the same though, but the numbers like 5 times higher for both when betagal is expressed.

using 0,5 µg of reporter and betagal, and 0,1 µg of expression vector with my receptor, for 500 000 HepG2 cells.

Greatful for any advice!! :-)

[Dr Teeth]

When you transfect in without b-gal, do you use an equimolar amount of a naked vector (vector without beta-gal) or do you just have the same transfection pool but without b-gal?  If you don't balance your two transfections on an equimolar level (not equal ug!), you should.  Also, there can be promoter induced effects (e.g squelching, etc.) if differing molar amounts of plasmids with different promoters are used.