I want to develop an assay platform for two genes in the same construct. Where GFP will be downstream to one of the gene and RFP for the other one. And finally I want to see the receptor mediated activity in terms of quantitative measurement of GFP and RFP just like the luciferase based reporter assays. Anyone have experience in such assays ?
If you just want promoter reporters you can buy them from GeneCopoeia. They will even make you any custom contruct you want: http://www.genecopoeia.com/product/promoter-reporter-clones/
Hi,
during my PhD, I did something similar, just luciferase-based. And it didn't work out at all. I got very robust results in luciferase assay but they turned out to be artefacts. So be sure to control at an early stage of your project that your results are not only robust but also relevant.
Good luck,
Cindy
Ratiometric analysis is always technically demanding. We use two very stable reporters (cytosolic GUS and secreted amylase) and we can normalise our results because both proteins are equally stable and can be detected with very robust colourimetric assays. In our latest publication, we describe the dual expression vector and how to do dose-response analysis ina ratiometric manner.
Gershlick, D., de Marcos Lousa, C., Foresti, O., Lee, A.J., Pereira, E.A., daSilva L.P. and Denecke,. (2014). Golgi-dependent transport of vacuolar sorting receptors is regulated by COPII, AP1, and AP4 protein complexes in tobacco. The Plant Cell Online 26 (3), 1308-1329
We have also worked with GFP and RFP, but we find that the two proteins show different bleaching rates, and that this depends on the cell compartment too. So depending on how long you image, you get completely different ratios and the reproducibility is insufficient for quantification.
Hi,
You may try to use ELISA based assay for quantitative GFP/RFP detection.
Best,
Lena
We perform an assay for GFP in liquid samples (even in bacterial or cell suspensions) using the Qubit phluorometer. It is quite reliable. A summary of the method is available in our paper by Lizier M (2010).
In my experience, the problem with the double labelling EGFP/RFP (T4) is the partial overlapping of emission and excitation spectra. Indeed blue light (470-480 nm) is able to excitate both. In addition most of emission filters are not so sharp in discriminating between the two emission wavelength.
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