To generate the GST or His fusion construct of AR-V7, we cloned PCR-amplified coding region of AR-V7 into the pGEX-6p-1 or pET-28a vector (Millipore Sigma). These constructs were transformed individually into E. coli strain BL-21, and protein expression was induced using 0.4 mM IPTG. Fusion proteins were purified using glutathione-agarose beads (GE Life Sciences) or Ni-NTA resin (Thermo Fisher). For pull-down assays, 2 μg of His-AR-V7 or AR-FL recombinant protein (Creative Biomart) was incubated with 2 μg of GST-AR-V7 protein, GST alone, or GST-fused catalytic domain of JMJD2B that was immobilized on glutathione beads in 50 μl of binding buffer (25mM Tris, 0.1% TX-100, 0.5 M NaCl in water, and proteinase inhibitor cocktail) for 2 hr at 4°C on a rotating platform. After centrifugation at 5000xg for 15 sec, the agarose beads were washed 4 times with the binding buffer. The proteins were then subjected to SDS-PAGE and Western blotting.
Ar fl recombinant protein
Manufactured by Creative Biomart
AR-FL recombinant protein is a laboratory reagent produced by Creative Biomart. It is a full-length, recombinant form of the AR (Androgen Receptor) protein. The core function of this product is to serve as a research tool for studying the structure, function, and interactions of the AR protein in various biological systems.
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2 protocols using ar fl recombinant protein
1
Recombinant AR-V7 Fusion Protein Generation
2
Recombinant AR-V7 Fusion Protein Generation
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To generate the GST or His fusion construct of AR-V7, we cloned PCR-amplified coding region of AR-V7 into the pGEX-6p-1 or pET-28a vector (Millipore Sigma). These constructs were transformed individually into E. coli strain BL-21, and protein expression was induced using 0.4 mM IPTG. Fusion proteins were purified using glutathione-agarose beads (GE Life Sciences) or Ni-NTA resin (Thermo Fisher). For pull-down assays, 2 μg of His-AR-V7 or AR-FL recombinant protein (Creative Biomart) was incubated with 2 μg of GST-AR-V7 protein, GST alone, or GST-fused catalytic domain of JMJD2B that was immobilized on glutathione beads in 50 μl of binding buffer (25mM Tris, 0.1% TX-100, 0.5 M NaCl in water, and proteinase inhibitor cocktail) for 2 hr at 4°C on a rotating platform. After centrifugation at 5000xg for 15 sec, the agarose beads were washed 4 times with the binding buffer. The proteins were then subjected to SDS-PAGE and Western blotting.
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