MG tumor cell pellets (at least 3 × 106 cells) or Gfi1-infected NIH-3T3 cells (ATCC CRL-1658) were resuspended in lysis buffer (150 mM NaCl, 20 mM Tris, 1% Triton X-100) with protease inhibitors (Roche, cat #1836153). Lysates were precleared for 30 min with Protein G agarose beads (Millipore #16-201D, Cell Signaling #37478). Totally, 10% of the sample was saved as input, and the rest was split in half for immunoprecipitation with experimental and control antibodies. Samples were incubated with antibodies specific for Gfi1 (1 µg, Santa Cruz sc-8558), Lsd1 (1 µg, Abcam ab17721) or CoREST (1 µg, Millipore cat #07-455), or with isotype control antibodies (1 µg, Santa Cruz sc-2028) for 1 h. Protein G beads were added to the samples and washed three times before preparing for Western blot by adding 4× sodium dodecyl sulfate (SDS) sample buffer and boiling.
Ab17721
Manufactured by Merck Group
Ab17721 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of this product is to facilitate various laboratory tasks and experiments. No further details on the intended use or specific applications are provided.
Automatically generated - may contain errors
3 protocols using ab17721
1
Gfi1 Protein Interactome Profiling
2
Immunoprecipitation of Nuclear Proteins
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Cell nuclear extract was pre-cleared with control IgG [Bioss (Boston, MA), bs-0295G-HRP] and protein A/G beads (Invitrogen, 10004D). Pre-cleared extract was then incubated with specific antibody (10 μg; LSD1, Abcam ab17721; GATA3, MILLIPORE 9076) or negative control IgG (Bioss, bs-0295G-HRP) overnight at 4 °C with rotation. Dynabeads (Invitrogen, 10004D) were added to the mixture and rotated for 3 h at 4 °C. After a brief centrifugation, the pellet was washed in RIPA buffer (1% DOC Sodium deoxgoholate, 0.1% SDS) 4-5 times at 4 °C for 10 min, and the immunoprecipitated protein complexes were analyzed by Western blot analysis with specific antibodies.
3
Immunoprecipitation of Nuclear Proteins
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