2 replies to this topic

[PaigeD]

Hey all.

I'm working on a luciferase expression assay using a protein of interest with different promoter-reporter constructs. I thought that the best cell line to do the assays in would be one without endogenous proteins of interest. However, a colleague told me that a relevant cell line, even if it has endogenous protein, would be best because it best recapitulates the necessary cofactors that my protein may need in order to induce expression from the promoter-reporters. I thought that having endogenous protien would conflict the results. What do you think is best??

Thanks,
Paige

[Dr Teeth]

PaigeD, on Apr 21 2009, 12:09 PM, said:

Hey all.

I'm working on a luciferase expression assay using a protein of interest with different promoter-reporter constructs. I thought that the best cell line to do the assays in would be one without endogenous proteins of interest. However, a colleague told me that a relevant cell line, even if it has endogenous protein, would be best because it best recapitulates the necessary cofactors that my protein may need in order to induce expression from the promoter-reporters. I thought that having endogenous protien would conflict the results. What do you think is best??

Thanks,
Paige

Don't use a cell line that endogenously expresses your protein of interest, unless you are just transfecting in reporters and not the protein itself. If you are just testing different promoter-luc constructs, endogenous expression of your transcription factor is fine unless you want to alter those levels as well.  In that case, the best cells to use would be cells that have been mutated or selected against expression of your desired protein.  This way you have all the necessary cofactors, but don't have a conflicting endogenous protein.  In either case, a test using multiple cell lines is the best.

Edited by Dr Teeth, 21 April 2009 - 11:01 AM.

[newarray]

First i would like to go with the cells which doesnt have endogenous expression of the protein. Even though all controls (like vector alone, promoter construct alone) are used with the cell line which expresses the endogenous protein it do bind to the promoter and results in some threshold expression where we will not get further differences in expression upon induction.

Hope this helps