Can anyone recommend a solid protocol for measuring GFP in S. cerevisiae whole cell suspensions using a plate reader? I could use some direction on normalizing, appropriate sample volumes and ODs, and standard curves if possible. Thank you!
I think I might be able to help you, but I'm not completely sure what your goal is here. You want to use the GFP as a readout of cell numbers? Is it endogenous GFP production or external labeling? Do you want to measure both fluorescence and absorbance, since you also mention ODs? Will you be measuring over a time course or is it an endpoint assay?
We measure GFP fluorescence of S. aureus by bottom reading of 100ul suspensions in 96-well plates with a transparent bottom. The GFP signal is linearly correlated with OD and CFU
I'm attempting to use GFP to readout the activity of a promoter construct driving endogenous GFP expression. To that end I'm only interested in fluoresence at various endpoints. With respect to OD I meant the OD600. Essentially I want to make sure that I don't add too many cells and end up outside the linear range of the reader - in your experience is this a valid concern? Thanks for your help!
I have been using the BioTek Synergy 2 for performing OD600 readings to do growth curve measurements. I use Nunc Edge Sterile plates and use 100ul of media per well. Likely you will need a different plate that is opaque (the Nunc plates are clear).
The Synergy2 is modular, so you need to ensure that yours is equipped to perform OD 600 absorbance as well as GFP fluorescence.
Hope that helps, feel free to ask me any other questions
Thanks Max, just got a wonderfully linear OD600 curve of serial dilutions out of the reader. Cheers!
I guess the only solution is to measure those same serial dilutions again for GFP fluorescence. Our S aureus is very bright in GFP expression and in our assay we do sometimes reach a value above the maximum value our reader can measure, but that is only after extensive bacterial growth and in cases where the sensitivity of the detector was set too high. But this of course all depends on your reader and the expression per cell.
Indeed you want to have plates with black walls to prevent signal leaking from one well to the next.
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