I'm working on some samples being imaged in a confocal microscope and I want to know if GFP is stable enough to still be detected after cell death. My samples are showing a decrease in intensity for every cell in sample 1 and in sample 2, they are showing either complete quencing or full brightness for the other treatment.
Thanks,
Matt
Considering the fact the GFP (as many fluorescent protein) is pH-dependant and overally environment dependant, I think that it depends on its environment. Never asked myself this question, but I am following this, so if you find some more precise informations, could you post them here ?
What is the nature of cell death? If it is apoptosis, your GFP protein could be digested, precipitated or denatured. Also, keep in mind that dead cells are leaky, meaning your GFP might slowly diffuse.
The nature of cell death is unexplored. ROS levels tested so far are high in the mixed quenched sample. There is evidence of lipid peroxidation and absence or low DNA damage.
Hi
https://www.ncbi.nlm.nih.gov
Green fluorescent protein as a novel tool to measure apoptosis and necrosis.
Gfp will fade if u r fixing with paraformaldehyde,apart from that ,u can cross check the artifacts of microscope,sometime you may end up bleaching by over exposing, u can try low intensity of exitation laser,
Apart from that if you r expecting cell death by apoptosis or necrosis you can validate just make sure out positive control
Dear Matthew Winans , If you did a protein expression here by transfection method, Is it possible probably your expressions were not stronger here? In this case I would highly recommend you to review the protocol you are using for the protein expressions. also can you try the same experiment in a different cell line as well to verify that your protocol is robust for the expression? I believe you must be able to see the GFP signal even after the cell death.
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