The recombinant protein was created with a fusion of MBP in the PPR75641–756 N-terminus and 6xHis in the C-terminus, and was purified across two columns equipped with Ni2+ affinity resin (Ni-NTA Resin, GenScript) and MBP (PurKine MBP-Tag Dextrin Resin 6FF, Abbkine) in turn. The control fusion protein containing only MBP and His tags was purified as well. RNA probes (Supplementary Table S1 ) were synthesized and labeled with 6-FAM at the 5′ end by GenScript (Nanjing, China). For the RNA electrophoresis mobility shift assays (REMSAs), the dialyzed recombinant protein was incubated with 100 fmol RNA probes in a 10 × binding buffer (100 mM HEPES PH = 7.3, 200 mM KCl, 10 mM MgCl2, 10 mM DTT) condition and reacted in a 20 μl system for 30 min at 25°C with 10 units of RNasin, followed by separation on 8% native acrylamide gels in a 0.5× TBE buffer. After electrophoresis, the gels were screened using a Typhoon Trio Imager (GE).
Purkine mbp tag dextrin resin 6ff
Manufactured by Abbkine
PurKine MBP-Tag Dextrin Resin 6FF is a chromatography resin designed for the purification of recombinant proteins with MBP (Maltose-Binding Protein) tags. The resin is composed of cross-linked dextran beads that provide high capacity and selectivity for MBP-tagged proteins. The resin is suitable for large-scale purification and can be used in various chromatographic techniques.
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2 protocols using purkine mbp tag dextrin resin 6ff
1
Recombinant Protein Purification and REMSA
2
Purification and in vitro pull-down of TOPPs
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To express recombinant proteins in prokaryotic cells, the coding sequences of the TOPPs were cloned into the pGEX 4T-3 (GST tag) or pET28a (HIS tag) vector and transformed into E. coli strain Rosetta. GST-tagged proteins were purified with Glutathione Sepharose 4B beads (10250335, GE Healthcare) following the manufacturer's instructions. HIS tagged proteins were purified with BeaverBeads™ IDA-Nickel (17B026101, Beaver) following the manufacturer's instructions. Protein concentration was determined using a BCA protein assay kit (Solarbio, PC0021).
The in vitro pull-down assay was performed as described (Guo et al., 2015) . ATG13a-MBP and ATG13b-MBP were purified using PurKine™ MBP-Tag Dextrin Resin 6FF (BMR20206, Abbkine). MBP beads containing ATG13a-MBP and ATG13b-MBP were incubated with purified GST or TOPP4-GST protein at 4°C for 2 h with gentle shaking. After washing several times with wash buffer, the proteins were detected by immunoblotting with anti-GST antibody.
The in vitro pull-down assay was performed as described (Guo et al., 2015) . ATG13a-MBP and ATG13b-MBP were purified using PurKine™ MBP-Tag Dextrin Resin 6FF (BMR20206, Abbkine). MBP beads containing ATG13a-MBP and ATG13b-MBP were incubated with purified GST or TOPP4-GST protein at 4°C for 2 h with gentle shaking. After washing several times with wash buffer, the proteins were detected by immunoblotting with anti-GST antibody.
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