My group has noticed some finicky behavior from our mCherry-tagged fusion proteins. We are transiently transfecting HEK293 cells with fusions in which mCherry (~230 aa) is between an N-terminal Gal4 DNA-binding domain and a C-terminal transcription activation domain (VP64 and other types). We use the mCherry-fusions to enhance activation at a Gal4-luciferase transgenic chromosomal target gene.
24 hours after transfection, we can detect immediate strong activation of the luciferase target gene even when mCherry (RFP) is very dim or undetectable under the microscope and via flow cytometry. When we wait 48 - 72 hours after transfection, the mCherry signal becomes brighter...the intensity of the mCherry-fusion is changing over time, but the response from the luciferase target does not track with this change.
Our conclusion is that the mCherry-fusion is functional but for some reason not always detectable.
This is a problem because we want to use RFP to quantify the level of activator protein (to calculate activator efficacy as luciferase response per RFP units) without having to do Western blotting or ELISA.
Could oxidative stress in the cells or changes in the pH of the growth medium over time be affecting the fluorescence of mCherry?
Thank you for any advice you can offer.
"A characteristic parameter for estimating the rate, at which an FP becomes fluorescent, is maturation half-time, which is the time required for the FP to reach a half of its maximal fluorescence. The maturation half-time can range from minutes to hours (Table I)."
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987785/
Hi Karmella,
This is an intriguing problem, as mCherry is usually a pretty friendly FP. I wouldn't think that pH is factor here because the pKa of mCherry is
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology