Plasmids expressing wild-type or mutant Cdc19-strep constructs were transformed into E. coli cells (Rosetta). Cells were grown at 37 °C in LB media (1% peptone, 0.5% yeast extract, 0.5% NaCl) with 30 μg/ml chloramphenicol and 100 μg/ml carbenicillin to OD600 0.6. Protein production was induced by adding 0.1 mM IPTG and growing cells at 16 °C overnight. Cells were harvested by centrifugation, resuspended in pre-cooled lysis buffer (100 mM Tris/HCl pH 7.4, 200 mM NaCl, 1 mM MgCl2, 10% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor mix (Roche) and 75 U/ml of Pierce universal nuclease), and processed for lysis by freezer milling (SPEX SamplePrep 6870 Freezer/Mill; five cycles of 2 min cooling and 2 min grinding at setting 15 CPS). Extracts were cleared by centrifugation (4 °C, 30 min, 48000 g), and the supernatant was loaded on a Strep-Tactin Superflow Plus column (Qiagen) at 4 °C following the manufacturer’s instructions. Proteins were eluted using 2.5 mM D-desthiobiotin in purification buffer (100 mM Tris/HCl pH 7.4, 200 mM NaCl, 1 mM MgCl2, 10% glycerol, 1 mM DTT, 1 mM PMSF). Fractions were collected, checked by SDS–PAGE and Coomassie blue staining, and pure fractions were pooled and aliquoted for storage at −80 °C.
Strep tactin superflow plus column
Manufactured by Qiagen
The Strep-Tactin Superflow Plus column is a chromatography column designed for the purification of Strep-tagged proteins. It features a high-capacity Strep-Tactin resin and is suitable for medium to large-scale protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor mix, by Roche (2 mentions)
Sampleprep 6870 freezer mill, by SPEX (2 mentions)
Superdex 75 increase column, by GE Healthcare (1 mentions)
Resource q, by GE Healthcare (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
3 protocols using strep tactin superflow plus column
1
Purification of Cdc19 Protein Variants
2
Purification of Cdc19 Protein Variants
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Plasmids expressing wild-type or mutant Cdc19-strep constructs were transformed into E. coli cells (Rosetta). Cells were grown at 37 °C in LB media (1% peptone, 0.5% yeast extract, 0.5% NaCl) with 30 μg/ml chloramphenicol and 100 μg/ml carbenicillin to OD600 0.6. Protein production was induced by adding 0.1 mM IPTG and growing cells at 16 °C overnight. Cells were harvested by centrifugation, resuspended in pre-cooled lysis buffer (100 mM Tris/HCl pH 7.4, 200 mM NaCl, 1 mM MgCl2, 10% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor mix (Roche) and 75 U/ml of Pierce universal nuclease), and processed for lysis by freezer milling (SPEX SamplePrep 6870 Freezer/Mill; five cycles of 2 min cooling and 2 min grinding at setting 15 CPS). Extracts were cleared by centrifugation (4 °C, 30 min, 48000 g), and the supernatant was loaded on a Strep-Tactin Superflow Plus column (Qiagen) at 4 °C following the manufacturer’s instructions. Proteins were eluted using 2.5 mM D-desthiobiotin in purification buffer (100 mM Tris/HCl pH 7.4, 200 mM NaCl, 1 mM MgCl2, 10% glycerol, 1 mM DTT, 1 mM PMSF). Fractions were collected, checked by SDS–PAGE and Coomassie blue staining, and pure fractions were pooled and aliquoted for storage at −80 °C.
3
Purification of Mad1 C-Terminal Domain
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The coding region of Mad1597–719 was cloned by USER® (NEB) into a modified pRSFDuet‐1 vector (71341‐3, Sigma‐Aldrich) with an N‐terminal double Strep His6‐tag, followed by a tobacco etch virus (TEV) protease cleavage site (Demple & Linn, 1982 (link); Bitinaite et al,1992 ). Mad1 mutants were generated using the QuikChange™ Lightning Site‐Directed Mutagenesis Kit (Agilent), developed by Stratagene Inc. (La Jolla, CA) (Nøhr & Kristiansen, 2003 (link)). Mad1 constructs were transformed into Rosetta™ 2 (DE3) Singles™ Competent Cells (71400, Novagen) for expression. Expression was induced with 0.5 mM IPTG and grown overnight at 18°C. Cells were lysed in 25 mM HEPES pH 8.1, 250 mM NaCl, 2 mM DTT, 5% glycerol, 2 mM EDTA supplemented with lysozyme, and Complete™ EDTA‐free protease inhibitors (Roche). Proteins were purified over a Strep‐Tactin Superflow Plus column (QIAGEN) and cleaved with TEV protease overnight at 4°C. The cleaved Mad1CTD was then diluted to 50 mM NaCl, purified over an anion exchange (Resource Q, GE Healthcare) column, followed by size exclusion chromatography using a Superdex 75 Increase column (GE Healthcare). Mad1CTD was concentrated to 35 mg/ml in a buffer of 20 mM HEPES pH 7.5, 100 mM NaCl and 1 mM TCEP.
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