4 replies to this topic

[pras45]

Hi all,
I am using GFP tagged constructs for luciferase assay system. Is this a good idea? would it interfere with the reading? or I am better off using non-fluorescent tagged protein.

thanks in advance

[klmilum112]

pras45, on Mar 16 2010, 06:17 AM, said:

Hi all,
I am using GFP tagged constructs for luciferase assay system. Is this a good idea? would it interfere with the reading? or I am better off using non-fluorescent tagged protein.

thanks in advance

When used the luciferase assay system, I just used a non-fluorescent tagged protein. I'm not sure it would interfere with the reading, but it's better to be safe than sorry. Are you using the GFP construct as a internal control? If that is the case, you might want to use the dual luciferase system in which you can use Renilla.

[Dr Teeth]

pras45, on Mar 16 2010, 10:17 AM, said:

Hi all,
I am using GFP tagged constructs for luciferase assay system. Is this a good idea? would it interfere with the reading? or I am better off using non-fluorescent tagged protein.

thanks in advance

I used GFP expression in a dual-reporter study and its expression did not influence the luciferase activity in any way, nor do I think it should.  Although the emission spectrum for luminescence is  often similar to that for fluorescence, the manner in which the emission is achieved differs.  For luminescence, photons result from energy transitions from excited-state molecular orbitals to lower energy orbitals stemming from the product of exothermic chemical reactions.  In contrast, photons are generated during fluorescence by the absorption of light by a compound followed by emission of light at a lower energy from the excited state of the molecule.

[KevinK]

I agree with Dr. Teeth.  The presence of GFP in the sample should not be a concern.  When yuo say the construct is GFP tagged, though, oif you are introducing a transcription facotr for instance and using luciferase to measure the activity of the transcription factor, you will want to verify that you get equivalent results with both the tagged and untagged versionof the protein.  Hope this helps.

Kevin

[pras45]

Thanks for all your suggestions....