I have one protocol which has SDS in the spreading buffer, but I'm afraid that this will solubilise the proteins and cause them to dissociate with DNA. I would ideally like to look at DNA damage response proteins such as gH2AX in spread chromatin.
Look here, maybe it could help:
The macrosatellite DXZ4 mediates CTCF-dependent long-range intrachromosomal interactions on the human inactive X chromosome.
Andrea H Horakova, Shawn C Moseley, Christine R McLaughlin, Deanna C Tremblay, Brian P Chadwick
What do you mean by chromatin spread? Interphasic nuclei, metaphases? DNA fibres? What cell line? If you want to analyse interphasic nuclei and you work with lymphocytes you can use poly-l-lysine slides or spread the cells in normal slides using a cytospin. For adherent cells you just need to grow the cells in cover-slips or chamberslides. However, if you want to analyse metaphases, you should spread the cells in normal slides using a cytospin. Regarding the immunofluorescence protocol, you can check these papers:
Genetics and Molecular Biology, 23, 4, 1107-1114 (2000)
Immunofluorescence in cytogenetic analysis: method and applications
Jeppesen P.
PLoS One. 2011;6(9):e25113. doi: 10.1371/journal.pone.0025113. Epub 2011 Sep 23.
Gamma-H2AX-based dose estimation for whole and partial body radiation exposure.
Horn S, Barnard S, Rothkamm K.
Genes Chromosomes Cancer. 2009 Sep;48(9):745-59. doi: 10.1002/gcc.20679.
Breaks invisible to the DNA damage response machinery accumulate in ATM-deficient cells.
Martín M, Terradas M, Iliakis G, Tusell L, Genescà A.
Hello, thank you for your responses.
By chromatin spread, I mean chromatin that has been isolated and spread from lysed nuclei. Briefly, the protocol I'm using is to isolate the nuclei from mouse fibroblasts, and once I have isolated nuclei, lyse these to release the chromatin, and then using a cytocentrifuge this isolated chromatin is spread onto microscope slide.
The problem I have is that in order to lyse the cells, SDS is used, which will denature the protein content of the nucleus, which is good for getting relaxed chromatin, but consequently impossible to look at proteins associated with DNA.
I was wondering if there is a protocol that is more gentle that can isolate chromatin but allow the retention of proteins, maybe a non-ionic detergent. I considered gently fixing the nuclei first, but I believe that this would not allow relaxed chromatin.
Hopefully there is a goldilocks zone where I can isolate relaxed chromatin but still retain proteins.
Any suggestions would be greatly welcomed.
Thank you,
Rhys
I have no expertise in chromatin isolation, so I can't help you. I'm sorry. But I think of trying TritonX100. Good luck!
Finally did you find a protocol this? I would like to do the same. So please tell me how it works for you.
Thanks
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