Nature research paper: Cooperation between bHLH transcription factors and histones for DNA access
containing an N-terminal Strep II tag. An additional N-terminal EGFP tag and C-terminal sortase-6×His tag were fused in-frame to improve purification. GFP–OCT4 was expressed in 4-l cultures ofHigh Five cells using the Bac-to-Bac system . Cells were cultured at 27 °C, collected two days after infection, resuspended in lysis buffer and 250 μM TCEP) and lysed by sonication.
. In general, 1–2 l of CLOCK-BMAL1 bHLH-PAS-AB-expressing insect cells were pelleted and resuspended in His buffer A glycerol, 0.1% Triton X-100 and 5 mM β-mercaptoethanol). Cells were lysed by cell disruption and subsequent sonication for 3 min . Lysate was clarified by centrifugation at 45,000 rpm for 45 min. Ni-NTA affinity purification was performed on a 5 ml HisTrap FF . After 14-column washes in His buffer A, the column was further washed with 6.
, with some modifications. First, cell pellets were resuspended in hypotonic buffer and homogenized. The homogenate was then centrifuged and the packed nuclear volume was determined. The pellet was resuspended in 2 pnv of pre-extraction buffer and the suspension was centrifuged . The pellet was then resuspended in 0.5 pnv of low-salt buffer , followed by the dropwise addition of 0.5 pnv of high-salt buffer .
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