Technical Reference #1940

1940.

The Interaction of NSBP1/HMGN5 with Nucleosomes in Euchromatin Counteracts Linker Histone-Mediated Chromatin Compaction and Modulates Transcription Mark Rochman; Yuri Postnikov; Sarah Correll; Cedric Malicet; Stephen Wincovitch; Tatiana S. Karpova; James G. McNally; Xiaolin Wu; Nina A. Bubunenko; Sergei Grigoryev; and Michael Bustin, National Cancer Institute, Molecular Cell, 35(1940), (2009)
Link To Paper

Materials & Methods:
For live cell imaging cells were plated on MatTek glass-bottom culture dishes 24 hr before transfection and constant temperature of 37 C was maintained during the experiment. For fixed cells imaging cells were plated on poly-Dlysine- coated coverslips on 6-well plates. Cells were fixed by freshly prepared 4% paraformaldehyde for 5–15 min at RT and then either permeabilized by 0.5% Triton X-100 for 5 min at RT or postfixed in cold methanol for 5 min at ÿ20 C. Following fixation cells were blocked in 1% BSA for 30 min at RT and incubated with indicated primary and secondary Abs for 1 hr at RT. Standard dilution for primary and secondary antibodies was 1:200 in blocking buffer. DNA was counterstained with Hoechst dye. Confocal images were collected using a Zeiss LSM 510 system mounted on a Zeiss Axiovert 200M microscope (Carl Zeiss Inc.; Thornwood NY) using an oil-immersion Plan-Apochromat 633/1.4 DIC objective lens. Colocalization of proteins with DNA was analyzed by the ImageJ program using the Colocalization Finder Plugin.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
SV-40-transformed MEFs