Hi Gita,

Surely you can measure GFP and RFP in beta-carotene expressing cells. Good you mentioned the name of GFP and RFP variants. Among these the main concern is whether spectra of beta-carotene overlaps with AcGFP? No it does not. Ex/Em of AcGFP is 475/505nm and that of beta-carotene Ex ranges from 527 to 540nm in different organic solvent. You can use narrow pass filters in your Flow to cut out any signal beyond 520nm. DsRED is not overlapping with the beta-carotene so it is of least concern to you in this case.

Best!

Hi Gita, the only problem I see with your idea is with the laser/filters setup of the flow cytometer. You will need both a blue (488) and a green (561) laser to properly detect acGFP and dsRed, respectively. Usually the green laser is mounted as an alternative to the blue one, which is by far the most commonly used. So you will need a very special configuration in your flow to be able to detect both molecules. Check with your flow core facility or whomever runs the cytometer there to know the details of your instrument.

Alternatively, you could use an anti-dsRed antibody conjugated to APC or something else that you can easily detect with your instrument.

Good luck

Andrea Galli Ankur Bothra

Hi Andrea,

Thanks a lot for your answer!

I produced these three proteins in yeast Saccharomyces cerevisiae cells. Cells diluted in water are used for the measurement using BD FACSCalibur Flow Cytometer with a 488 nm excitation laser and 530/30 nm band pass filter for detection (not 520 nm). The program to distinguish AcGFP1 and DsRED was established and so I can measure it using our FACSCalibur. The problem is the possible overlap between beta-carotene and AcGFP1. Actually, FCASCalibur is not good choice. Ankur suggested trying other Flow machines and calculating the Ex/Em maxima for beta-carotene and AcGFP1 in my system, first. I am now working on it.