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problems at normalizing luciferase assay data - (Jul/08/2011 )

Hello All,

I have been trying to interpret my Luciferase assay data, my intracelluar Leucine-zipper protein of interest is attached with Gaussia luciferase (secretion signal deleted. after 48 hours of transfection to the H4 cell, I measured the luciferase activity in the culture media and the cell lysate. to my surprise 85% of the activity are in the Culture media (CM) and 15% are in the cell lysate. below is my data

Untransfected lysates reading: 300 units
Zip1+Zip2 lysate reading: 1134329 units
20ul of sample was assayed (total lysate was 100ul and lysates
were prepared according to the protocol)

Untransfected CM reading: 25292 units
Zip1+Zip2 CM reading: 1077232 units
20ul of sample was assayed (total CM was 500ul)

as you can see Total Z1+Z2 lysate activity should be 5671645

because the CM has serum in it, the untransfected CM's
background noise was very high, So I did 1077232-25292=1051940
and multiply the number by 25 (for a total activity in 500ul)
which gives me 26298500.

Based on the above calculation, 80% of the signal are outside
of the cell which worries me a lots.

However when I divide my untransfeced CM reading by my
untransfected lysates reading: 25292/300= ~85 fold and taking
the 85 fold in to the lysates reading, there are only 5%
activity found in the CM. Perhaps X amount of serum give me
300 units and Y amount of serum would give me 25292 units of

I am wondering if the division method is legitimate to normalize my assay?



Why would you read the activity in your cell culture medium?