2 replies to this topic

[sitare]

Hi everybody;

I have a problem about my luc assay. I m trying to co-transfect mda mb 231 cell line.Inittially,i used tk renilla luc for normalization. After 48 hour transfection, i saw tk renilla luc values were very very high compared firefly luc values.
Is this normal? Or if this is a problem, what can i do to solve this problem?

Thanks a lot

[96well]

Hi,
it is usual to have Renilla values 2-4 orders of magnitude higher than the Firefly's. This is somewhat expected in Firefly/Renilla ration of 1:1 (the tk-renilla has a strong promoter to strongly drive renilla expression without influence of the signaling pathways you could modulate during your treatments. The firefly promoter is quite the opposite: usually a weak promoter with some responsive elements to 'report' the activity of the pathway you are interested in).

If you are worried about the high Renilla values you can use less Renilla plasmid.

[sitare]

96well, on 16 October 2010 - 12:04 PM, said:

Hi,
it is usual to have Renilla values 2-4 orders of magnitude higher than the Firefly's. This is somewhat expected in Firefly/Renilla ration of 1:1 (the tk-renilla has a strong promoter to strongly drive renilla expression without influence of the signaling pathways you could modulate during your treatments. The firefly promoter is quite the opposite: usually a weak promoter with some responsive elements to 'report' the activity of the pathway you are interested in).

If you are worried about the high Renilla values you can use less Renilla plasmid.

thanks a lot 96 well:)