N-Terminal strep-6xHis-TEV mTagBFP2 RanQ69L was cloned into the pST50 vector via Gibson Assembly (New England Biolabs). The plasmid was transformed into E. coli Rosetta2 cells (EMD Millipore) for protein expression. Cells (4 L) were grown until OD600 = 0.8 at 37 °C in LB media, and then protein expression was induced using 500 mM IPTG for 18 hours at 16 °C before cells were pelleted. RanQ69L was purified in the following manner:24 (link),25 (link) we lysed cells by using an Emulsiflex french press (Avestin) in binding buffer (100 mM Tris–HCl, 450 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 2.5 mM PMSF, 6 mM BME, pH 8.75). We centrifuged the lysate at 20 000 × g and loaded the supernatant onto a StrepTrap HP column (GE Healthcare, 5 ml). Protein was eluted in binding buffer with 2.5 mM d -desthiobiotin, and dialyzed overnight into CSF-XB buffer (10 mM HEPES, 100 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10% sucrose w/v, pH 7.7). We included 200 μM GTP in lysis and elution buffers and obtained about 0.5 ml of 200 μM RanQ69L.
E coli rosetta 2 cells
Manufactured by Merck Group
The E. coli Rosetta 2 cells are a specialized bacterial strain used for protein expression. They are designed to enhance the expression of eukaryotic proteins that contain rare codons in E. coli. The Rosetta 2 strain provides tRNAs that are rare in E. coli, allowing for improved translation of proteins that would otherwise be poorly expressed.
Automatically generated - may contain errors
5 protocols using e coli rosetta 2 cells
1
Purification of Mutant Ran GTPase
2
Purification of Recombinant ZAP-S Protein
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Recombinant ZAP-S N-terminally tagged with 6×His-SUMO was purified from E. coli Rosetta 2 cells (Merck) by induction with 0.2 mM isopropyl β-d-1-thiogalactopyranoside for 18 h at 18 °C. Cells were collected, resuspended in lysis buffer (50 mM HEPES/KOH pH 7.6, 1 M NaCl, 1 mM DTT, 1 mM PMSF) and lysed in a pressure cell. The lysate was cleared by centrifugation and ZAP-S was captured using Ni-NTA resin (Macherey-Nagel). After elution with 500 mM imidazole, ZAP-S was further purified and the bound nucleic acids removed by size exclusion chromatography (HiLoad® 16/600 Superdex® 200) in 20 mM HEPES/KOH pH 7.6, 1 M KCl, 1 mM DTT, 20% glycerol. Protein identity was verified by SDS-PAGE as well as western blotting (Supplementary Fig. 2D ). Purified ZAP-S was rapidly frozen and stored in aliquots at −80 °C. His-SUMO IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany).
3
Purification of RanQ69L Protein
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N-terminal strep-6xHis-TEV mTagBFP2 RanQ69L was cloned into the pST50 vector via Gibson Assembly (New England Biolabs). The plasmid was transformed into E. coli Rosetta2 cells (EMD Millipore) for protein expression. Cells were grown until OD600= 0.8 at 37 °C in LB media, and then protein expression was induced using 500 mM IPTG for 18 hours at 16 °C before cells were pelleted.
RanQ69L was purified mostly as described (21, 22) . Briefly, cells were lysed using an Emulsiflex french press (Avestin) in binding buffer (100 mM Tris-HCl, 450 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 2.5 mM PMSF, 6 mM BME, pH 8.75). The lysate was centrifuged at 20,000 x g and the supernatant was loaded onto a StrepTrap HP column (GE Healthcare). Protein was eluted in binding buffer with 2.5 mM D-desthiobiotin, and dialyzed overnight into CSF-XB buffer (10 mM HEPES, 100 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10% sucrose w/v, pH 7.7). 200 μM GTP was included in lysis and elution buffers.
RanQ69L was purified mostly as described (21, 22) . Briefly, cells were lysed using an Emulsiflex french press (Avestin) in binding buffer (100 mM Tris-HCl, 450 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 2.5 mM PMSF, 6 mM BME, pH 8.75). The lysate was centrifuged at 20,000 x g and the supernatant was loaded onto a StrepTrap HP column (GE Healthcare). Protein was eluted in binding buffer with 2.5 mM D-desthiobiotin, and dialyzed overnight into CSF-XB buffer (10 mM HEPES, 100 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10% sucrose w/v, pH 7.7). 200 μM GTP was included in lysis and elution buffers.
4
Purification of Recombinant ZAP-S Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant ZAP-S N-terminally tagged with 6×His-SUMO was puri ed from E. coli Rosetta 2 cells (Merck) by induction with 0.2 mM isopropyl β-d-1-thiogalactopyranoside for 18 h at 18°C. Cells were collected, resuspended in lysis buffer (50 mM HEPES/KOH pH 7.6, 1 M NaCl, 1 mM DTT, 1 mM PMSF) and lysed in a pressure cell. The lysate was cleared by centrifugation and ZAP-S was captured using Ni-NTA resin (Macherey-Nagel). After elution with 500 mM imidazole, ZAP-S was further puri ed and the bound nucleic acids removed by size exclusion chromatography (HiLoad® 16/600 Superdex® 200) in 20 mM HEPES/KOH pH 7.6, 1 M KCl, 1 mM DTT, 20% glycerol. Protein identity was veri ed by SDS-PAGE as well as western blotting (Supplementary Fig. 2D). Puri ed ZAP-S was rapidly frozen and stored in aliquots at -80°C.
His-SUMO-IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany)
His-SUMO-IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany)
5
Purification of ZAP-S Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant ZAP-S N-terminally tagged with 6×His-SUMO was purified from E. coli Rosetta 2 cells (Merck) by induction with 0.2 mM isopropyl β-d-1-thiogalactopyranoside for 18 h at 18 °C. Cells were collected, resuspended in lysis buffer (50 mM HEPES/KOH pH 7.6, 1 M NaCl, 1 mM DTT, 1 mM PMSF) and lysed in a pressure cell. The lysate was cleared by centrifugation and ZAP-S was captured using Ni-NTA resin (Macherey-Nagel). After elution with 500 mM imidazole, ZAP-S was further purified and the bound nucleic acids removed by size exclusion chromatography (HiLoad® 16/600 Superdex® 200) in 20 mM HEPES/KOH pH 7.6, 1 M KCl, 1 mM DTT, 20% glycerol. Protein identity was verified by SDS-PAGE as well as western blotting (Supplementary Fig. 2D). Purified ZAP-S was rapidly frozen and stored in aliquots at -80 °C.
His-SUMO-IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany)
His-SUMO-IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany)
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