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Preprint

In vitro pull-down assay

YX Yue Xiong
YX Yan-ping Xu

Last updated date: Sep 25, 2022 Views: 555 Forks: 0

original An abbreviated version of this protocol was published in Science Advances in Sep, 2020
USP15 suppresses tumor immunity via deubiquitylation and inactivation of TET2
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  1. Biotin labeled single-stranded oligonucleotides used for TET-DNA binding assay were synthesized at Sigma, sequence as follows: 5'-GTATGCCTCATGCCGGACTTAACTGCAGTG-3' , 3'-CATACGGAGTACGGCCTGAATTGACGTCAC-5’.
  2. Two nanomole of complementary single-stranded oligonucleotides were mixed in annealing buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA), boiled for 5 min, transferred to a water preheated to 90 °C and gradually cooled overnight.
  3. Purify the Flag-TET2 protein from bacteria or cultured cells.
  4. The 1ng annealed DNA was immobilized to 10ul streptavidin beads by incubation together at 4°C for 2 hours and washed three times. Wahing buffer: 0.1% NP-40, 50 mM tris (pH 7.5), 150 mM NaCl.
  5. The 500ng purified protein Flag-TET2 was incubated with immobilized DNA at 4°C for 3 hours and washed three times. 
  6. The binding efficiency of TET2-DNA was determined by Western blot.
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Xiong, Y and Xu, Y(2022). In vitro pull-down assay. Bio-protocol Preprint. bio-protocol.org/prep1964.
  2. Chen, L., Smith, M. D., Lv, L., Nakagawa, T., Li, Z., Sun, S., Brown, N. G., Xiong, Y. and Xu, Y.(2020). USP15 suppresses tumor immunity via deubiquitylation and inactivation of TET2 . Science Advances 6(38). DOI: 10.1126/sciadv.abc9730

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