Strain PK14639 (PK14623 carrying pPK14637 plasmid) was grown in 1 L of Terrific broth (RPI) with 50 μg/mL ampicillin and 10 mM arabinose, inoculated to 0.5% from an overnight culture to an OD600nm of 1.0, and induced with 0.4 μM IPTG along with the addition of 2 mM cysteine and 0.2 mg/mL of Ferric ammonium citrate. After 1 h, the culture was transferred to 4°C and sparged with argon overnight. All subsequent steps were performed under anaerobic conditions in an anaerobic chamber (Coy Laboratory Products). The cells were harvested, resuspended in lysis buffer (50 mM potassium phosphate buffer, 100 mM NaCl, 10% glycerol, 1 mM DTT, pH 7.2), and lysed using a French press at 20,000 lb/in2. The lysate was centrifuged for 1 h at 4°C at 45,000 rpm in a Beckman 70.1 Ti rotor. The supernatent was then loaded onto a 5-mL StrepTrap HP (Cytiva) column equilibrated in the same lysis buffer and connected to a FPLC AKTA Pure system. The protein was eluted using a step gradient with lysis buffer containing 2.5 mM desthiobiotin (EMD Millipore). For measurement of the visible spectrum, the N terminus tagged protein was transferred under anaerobic conditions into a screw top quartz cuvette (Starna). The spectrum was recorded in a Perkin-Elmer λ25 spectrophotometer.
Streptrap hp
Manufactured by Cytiva
The StrepTrap HP is a chromatography column designed for the purification of proteins with a Streptag II affinity tag. It is composed of a high-performance agarose matrix with immobilized streptavidin, which enables the efficient capture and purification of Streptag II-tagged proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
λ25 spectrophotometer, by PerkinElmer (1 mentions)
70.1 ti rotor, by Beckman Coulter (1 mentions)
Anaerobic chamber, by Coy Laboratory Products (1 mentions)
293fectin, by Thermo Fisher Scientific (1 mentions)
Superdex 200 column, by Cytiva (1 mentions)
Midiprep kit, by Qiagen (1 mentions)
Pierce bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Overnight express autoinduction system 1, by Merck Group (1 mentions)
Histrap hp, by Cytiva (1 mentions)
Sonicator s 4000, by Bioventus (1 mentions)
4 protocols using streptrap hp
1
Production and Purification of Cysteine-Containing Protein
2
Purification of Coronavirus Proteins
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The coronavirus proteins were produced and purified as previously described (15 (link), 48 (link), 49 (link), 50 (link)). Plasmids encoding N-terminal domain proteins were obtained from GenScript. Plasmids were transiently co-transfected in FreeStyle 293-F cells using 293Fectin (ThermoFisher). All cells were tested monthly for mycoplasma. DNA was prepared using a Midiprep kit (Qiagen). On day 5 or 6 post transfection cell culture supernatants were clarified by centrifugation and filtration with a 0.8-μm filter. Stepwise purification included affinity chromatography using StrepTrap HP (Cytiva) ran in 1X Buffer W (IBA Lifesciences) and eluting in 1X Buffer E (IBA Lifesciences), and by size-exclusion chromatography using Superdex 200 columns (Cytiva) in 10 mM Tris pH8, 500 mM NaCl.
3
Purification of HA Proteins from E. coli
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Rosetta2 (DE3) Escherichia coli cells (Merck) were grown in Terrific broth medium. The expression of HA proteins was induced using Overnight Express Autoinduction System 1 (Merck) at 18 °C for 48 h. Cells were harvested and lysed in PBS (pH 7.4) by sonication. His-HA1-FLAG, His-HA2, and His-NanoHA were purified using HisTrap HP (Cytiva). Strep-tag II-tagged proteins were purified using StrepTrap HP (Cytiva). These column purification steps were performed following the manufacturer’s protocols. All proteins were dialyzed against PBS (pH 7.4) and stored at −80 °C until further analysis. The protein concentration of the samples was determined using the Pierce bicinchoninic acid assay (Thermo Fisher Scientific).
4
Purification of SUV39H2 Methyltransferase
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pACE1_SUV39H2 plasmid was transformed into ArcticExpress (DE3) competent cells (Agilent). Colonies were expanded in LB overnight and a 1 L culture grown at 37 °C to OD600 = 0.4 before induction with 1 mM IPTG for 24 h at 10 °C. The cultures were harvested by centrifugation at 3000×g and the pellets stored at − 80 °C.
The harvested bacterial cell pellets were resuspended in 10 × pellet volume of lysis buffer (25 mM Tris [pH 7.6], 300 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.5% NP-40) and subjected to freeze–thaw and lysozyme treatment as described above. The lysate was sonicated (3 cycles of 30 s on, 30 s off, on ice, with mixing by gentle inversion in between) at an amplitude of 40 using a Misonix S-4000 Sonicator, and centrifuged at 25,000×g for 30 min at 4 °C to remove insoluble material.
Affinity purifications for StrepII-SUV39H2 were performed with StrepTrap HP (Cytiva). The lysate was bound onto the column, which was washed 2.5 × column volumes wash buffer 1 (20 mM Tris [pH 7.6], 100 mM NaCl, 0.2 mM EDTA, 20% glycerol), 2.5 × column volumes wash buffer 2 (20 mM Tris [pH 7.6], 300 mM NaCl, 0.2 mM EDTA, 20% glycerol, 0.05% NP-40) followed by 2.5 column volumes wash buffer 1. Protein was eluted in wash buffer 1 supplemented with 2.5 mM desthiobiotin. Elution fractions containing SUV39H2 were dialyzed against BC100 with 20% glycerol at 4 °C to remove desthiobiotin.
The harvested bacterial cell pellets were resuspended in 10 × pellet volume of lysis buffer (25 mM Tris [pH 7.6], 300 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.5% NP-40) and subjected to freeze–thaw and lysozyme treatment as described above. The lysate was sonicated (3 cycles of 30 s on, 30 s off, on ice, with mixing by gentle inversion in between) at an amplitude of 40 using a Misonix S-4000 Sonicator, and centrifuged at 25,000×g for 30 min at 4 °C to remove insoluble material.
Affinity purifications for StrepII-SUV39H2 were performed with StrepTrap HP (Cytiva). The lysate was bound onto the column, which was washed 2.5 × column volumes wash buffer 1 (20 mM Tris [pH 7.6], 100 mM NaCl, 0.2 mM EDTA, 20% glycerol), 2.5 × column volumes wash buffer 2 (20 mM Tris [pH 7.6], 300 mM NaCl, 0.2 mM EDTA, 20% glycerol, 0.05% NP-40) followed by 2.5 column volumes wash buffer 1. Protein was eluted in wash buffer 1 supplemented with 2.5 mM desthiobiotin. Elution fractions containing SUV39H2 were dialyzed against BC100 with 20% glycerol at 4 °C to remove desthiobiotin.
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