I'am trying a supercoiling plasmid assay to show histone chaperone activity. I want to see the supercoiling of the PBS sk (+) - previously treated with topoisomerase I to relax the plasmid- after adding core histones from Hela and a chaperone. The problem is I also saw the supercoiled band in all controls, i.e. when adding either histones or chaperone that should not happen. Any advice?
Hi! well it's hard to help without a picture. you can check Fig 2B of my paper where I did that exact same assay. let me know if you have more questions!
https://www.researchgate.net/publication/7503519_The_HIR_corepressor_complex_binds_to_nucleosomes_generating_a_distinct_proteinDNA_complex_resistant_to_remodeling_by_SWISNF?ev=srch_pub
Article The HIR corepressor complex binds to nucleosomes generating ...
Thank you Philippe! I cheched your paper and efectivelly you described the same assay...At the beginning, all conditions I used are the same as yours, but something is wrong in my assays...I saw the supercoiled band even without chaperone and histones...
You mean, fully supercoiled? do you see any totally relaxed plasmid in the first place? if no relaxed one at all, then your Topo is not working. I do see some slight supercoiling in the mock samples, but that's pretty normal.
Thank you Philippe, I can't see a totally relaxed plasmid after treating with topoisomerase I...I saw the relaxed and some slight supercoiled band...but after incubating at 37ºC the supercoiled band increased...even without histones or chaperone...My topo is new...I used 2.5 U to relaxe 200 ng PBS plasmid, as many protocols recommend...
One more question: Did you remove topoisomerase I after treating plasmids and before mixing with Chaperone/Histones?
Thank you!
No, you keep the topoisomerase during the reaction, so it keep relaxing the DNA while more nucleosome are assembled onto the plasmid. Upon removal of the histones at the end of the reaction, the constrained supercoiled will be regenerated on the plasmid and visiualized on the gel. Good luck! You need very good plasmid prep, the best is to do a Cesium chloride purification using centrifugation, but a good/clean prep of plasmid DNA should do it as well!
Thank you very much Philippe...
Did you stain your agarose gel with ethidium bromide before or after running? I read that ethidium bromide can modify the mobility of supercoiled and relaxed bands...
I used to staine before (I mean, while electrophoresis is running), but I would try run the gel and then add the ethidium...What do you think about? Even I read someone tried cloroquine instead ethidium bromide...
Sorry for the inconveniences and thank you in advance!
- Badges
- Science topic