Journal: bioRxiv
Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells
doi: 10.1101/2020.05.28.122184
Figure Lengend Snippet: (A) Chromatography scheme for purification of SCC-B from NT2 nuclear extracts (NT2 NE). NT2 NE is first subjected to ammonium sulfate precipitation (55% saturation) followed by a series of chromatographic columns including nickel affinity agarose (Ni-NTA), cation exchangers phosphocellulose (P11), heparin (Poros-HE), Mono S, anion exchanger Poros-HQ, hydroxyapatite (HAP), and gel filtration medium Superose 6. SCC-B activity segregates from SCC-A (Dyskerin (DKC1) RNP) at the Poros-HE step. (B) Input fraction containing SCC-B activity from the Poros-HE step (IN), flow-through (FT) and various salt-eluted Mono S fractions were assayed for their ability to stimulate OCT4/SOX2-dependent transcription from the human NANOG promoter template engineered with four extra copies of the oct-sox composite binding element (bottom). All reactions contain purified general transcription factors (GTFs), Pol II, OCT4, SOX2, and recombinant XPC and DKC1 complexes. (C) Fractions assayed in vitro transcription shown in (B) are separated on a 4-12% gradient polyacrylamide gel and stained with silver. Filled arrowhead indicates the predominant polypeptide at ~110 kDa that co-migrates with SCC-B transcriptional activity. The following figure supplement is available for : .
Article Snippet: Transcriptionally active Q0.3 fraction (0.32–0.4 M) were pooled and applied directly to hydroxyapatite (HAP) type II ceramic resin (Bio-Rad), washed first at 0.38 M, then lowered to 0.1 M KCl in 3 CV.
Techniques: Chromatography, Purification, Filtration, Activity Assay, Binding Assay, Recombinant, In Vitro, Staining
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