Luc assay normalisation - protein quantification for normalisation on Luc Assay (B-gal?) (Jun/01/2007 )

I am trying to do a Luc Assay and here on my lab people use to quantify protein for normalization of Luc activity.
If we have much more dead cells on one particular experimental condition than on the others, the Luc activity will be lower but the amount of protein will be similar once that the dead cells continue to contribute to the overall protein amount.

Does it make any sense for you?
Is it a real problem?
Does B-gal normalisation assay help solving this problem?

Thanks in advance for any tip

ruineves

That is absolutely a problem and is the reason co-transfection with control vectors was introduced, also if something affects the transfection efficiency well to well you will not see this in the protein analysis. I personally feel that this is an inappropriate way to control your experiment and can only be justified in extreme cases (where say co-transfection affects something so cannot be done) and even then extensive analysis would have to be done first to verify that the protein is a reasonable measure (almost impossible but if for some reason you HAD to this may be acceptable-in a best possible control scenerio) B-gal normalization does help when the B-gal vector is co-transfected, but why not use a renilla Luc construct as a control? Renilla would be more compatible with your current system...

HTH and good luck

Thanks for your reply. Good to discuss.

But, doing co-transfections we are assuming that the efficiency of transfection of both constructs is the same, isn’t it? And that might not be true, isn’t it?

On my case, I am transfecting an 8Kb vector on K562 cell line. As it is a reasonable big vector, is not difficult to imagine that the control vectors (like renilla one) are much smaller. Do I have to assume equal transfection efficiencies, or there is another way?

Maybe the best way would be to have both luciferases (firefly and renilla) on the same vector each with its own independent promoter, no? But... wouldn’t be an enormous vector?

Any suggestion?

Thanks in advance

Rui Neves

Yes, but look at it this way, really the assumption is that the efficiency doesn't change not that they are equivalent, ie: even if the renilla control has 50% efficiency and your construct has 30% efficiency then that should be the same for all wells where both are transfected, so that if in one well your renilla control is now 25% it is safe to assume that whatever affected renilla also affected your vector which would then be at 15% efficiency so it is a perfectly valid way to control... the real issue is say you are doing deletion constructs, what if the deletion is higher efficiency than the original construct so now you get 60% efficiency of the deletion which makes it look like there is more activity when there really isn't... to me this is the better reason to keep both controls on the same vector. (although it is typically assumed that the effect on efficiency between the different vectors (say with deletion constructs) is minimal and can be assumed to be equivalent)

HTH and good luck!