I am studying a protein and from imaging I can see that my protein is recruited to sites of DNA damage. I wish to UV irradiate HEK293 cells in culture prior to collection and analyses by RNA-seq and mass spectrometer. Does anyone have an idea of how to (protocol and instrumentation) UV irradiate cells in culture for such studies?
Eexpose cells to 10 mJ/cm2 of UV UV cross-linker
(Stratalinker) are good and put in your values (i.e. 10 mJ) and it will expose the plate for the appropriate period of time. The time duration is calculated according to the UV source lamp' strength. Please read our paper for more details:
Hepatitis B virus X protein impedes the DNA repair via its association with transcription factor, TFIIH
Article in BMC Microbiology 11:48 · March 2011
Impact Factor: 2.98 · DOI: 10.1186/1471-2180-11-48 · Source: PubMed
I would like to add that one needs to remove culture medium in order to treat cells with UV as noted by Ishtiaq. Removal of medium delivers the UV-light in reproducible manner to cells.
Dear Gerald,
Alternatively you can expose HEK293 cells to Hydrogen peroxide (H2O2) treatment which causes DNA damage to the cells.I hope you can study the protein recruitment to sites of DNA damage.
all the best
I think an important issue to consider is whether you are interested in acute or chronic changes. If acute, you want to plan your experiments to maybe collect the cells within 6 hours. If you want to see changes over time, that's a different issue. We have done similar experiments in the lab but with different cell lines-
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