Hi all,
Im performing FRAP experiments on markers of DNA damage response transiently transfected into the cells. I would like to generate DBSs in a region of interest with 405nm laser and measure the kinetics of the targets there.
I tried to irradiate cells without sensitize them but from IF for gH2AX there was a small amount of damage, even with the highest settings, and the accumulation did not happen at all.
I could use BrDU, but I am not sure if it is suitable for 405 nm laser, any suggestion?
Dear Paolo Fagherazzi , looking at BrDU UV-Vis spectrum, 405nm wouldn´t excite it at all, at least using a continuous wave laser, that is what you often has in your microscope. (May be multiphoton absorption could do the job, but just may be and at a huge cost).
Furthermore, several commercial sources are selling antibodies specific for BrDU, labeled with Alexa-Fluor 405, so the labeled antibody is linked to the BrDU position and by irradiation with a 405 nm laser the fluorophore is excited and therefore you can know where the BrDU is and how it is diffusing in your FRAP experiment.
Here you can find a link to BrDU spectrum:
https://spectrabase.com/compound/JwNVPkeagv5#J3YFFJKgsBP
and here a link to a commercial source of anti-BrDU labeled with alexa-fluor 405:
https://www.rndsystems.com/products/bromodeoxyuridine-brdu-alexa-fluor-405-conjugated-antibody-bu-1_ic7225v
Hope it helps.
You are wellcome Paolo Fagherazzi , I hope it helps you with your research. Good luck!
Best!
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