Hi! I'm doing a renilla luciferase assay with coelenterazine and HEK293T cells transfected with AP-1 renilla luciferase. I'm wondering if the cells need to be lysed prior to adding coelenterazine and measuring the luminescence. Coelenterazine is cell-permeable, so I'm wondering if/why the lysis step would be necessary.
I understand that the proteins would be more exposed--would the luminescence be not as great through the cell membrane if we didn't do the lysis?
No. The cells need not be lysed. Because the substrate will be anyhow reacted by the luciferase to give the signal.
For renilla luciferase, it should be lysed prior to the assay, although the substrate can enter into cells. The data will be stable and accurate once cells are lysed.
If you do not want to lyse the cell, the naturally secreted Gaussia princeps luciferase (Gluc) or Cypridina Luciferase (Cluc) can be recommended. This kind of luciferase will be more ultrasensitive for the quantitative assay.
Using the Promega Dual-Luciferase Reporter (firefly and renilla), which I think is a variant of your own protocol; the lyse process is required. Specifically, the Passive Lyse Buffer will be the one to add just after the PBS wash, the lysis buffer will generally release the cell from plate attachment. lysis can take up to 15 minutes... you can centrifuge briefly at 10,000g to increase the lysis process.
Thankyou Dejun Ma and Olatunde Omotoso for your replies.
But won't lysing the cells affect the exact parameter of 'viability' that one wants to analyse.
Sindhu Kondath ... Dual Luciferase assay is not to test cell viability. among other things, it is mainly used to test DNA repair capability. For cell viability, I think MTT or WST-1 would be the best assay
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