M. acetivorans cells were harvested at an OD600 of 0.8 by anaerobic centrifugation (8000×g for 10 min at 4 °C) and the pellets were stored at -80ºC. For purification of recombinant protein, all the steps were carried out in an anaerobic chamber (Coy Laboratories) containing 95% N2 and 5% H2. The cells were thawed and resuspended in buffer NP (50 mM NaH2PO4 and 300 mM NaCl, pH 8.0) containing 4 μg/mL DNase I and 1 mM Benzamidine. The cells were sonicated 3 times followed by centrifugation (8000×g for 10 min at 4 °C). The cell lysate was loaded on a chromatography column containing 1 mL of Strep-Tactin superflow plus resin (Qiagen) pre-equilibrated with 12 mL of buffer NP. The column was washed with 5 ml buffer NP. Protein was eluted in 3 mL buffer NP containing 2.5 mM desthiobiotin (NPD) and stored at − 80 °C. The protein concentration was determined using the Bio-Rad protein assay42 (link). The purified protein was analyzed by 12% SDS-PAGE gel stained with Coomassie blue solution followed by de-staining. The protein bands were excised, trypsin digested, and sent for mass spectrometry (The University of Arkansas Statewide Mass Spectrometry Facility). UV–visible absorption spectrum was recorded using a Cary 60 spectrophotometer (Agilent Technologies) under anaerobic conditions.
Strep tactin superflow plus resin
Manufactured by Qiagen
Sourced in Germany
The Strep-Tactin Superflow Plus resin is a chromatography resin used for the purification of recombinant proteins. It is designed to bind to the Strep-tag II affinity tag, which is commonly used for protein purification. The resin provides high-performance binding and elution capabilities, allowing for efficient and selective protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Desthiobiotin, by Merck Group (2 mentions)
Protein assay, by Bio-Rad (1 mentions)
Cary 60 spectrophotometer, by Agilent Technologies (1 mentions)
Ni nta superflow resin, by Qiagen (1 mentions)
Centricon centrifugal filter units, by Merck Group (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Akta pure, by GE Healthcare (1 mentions)
Ezblue protein stain, by Merck Group (1 mentions)
Talon cellthru co2 chelating resin, by Takara Bio (1 mentions)
Desthiobiotin, by IBA Lifesciences (1 mentions)
Econo column chromatography column, by Bio-Rad (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Vivaspin, by Sartorius (1 mentions)
7 protocols using strep tactin superflow plus resin
1
Purification and Characterization of Recombinant Protein from M. acetivorans
2
Purification of CAK Complexes
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CAK complexes were purified using Nickel affinity, Strep-Tactin affinity, and size exclusion chromatography. Cells were resuspended in lysis buffer (250 mM KCl, 40 mM Hepes-KOH pH 7.9, 5 mM MgCl2, 5 mM β-mercaptoethanol, 10 mM imidazole, and 10% [vol/vol] glycerol supplemented with protease inhibitors and DNaseI) and homogenized by sonication. The lysate was cleared using a JA-20 rotor (18,000 rpm for 30 min at 4 °C) and incubated with Ni-NTA superflow resin (Qiagen) for 30 min. The beads were washed in purification buffer (250 mM KCl, 25 mM Hepes-KOH pH 7.9, 5 mM MgCl2, 5 mM β-mercaptoethanol, and 10% [vol/vol] glycerol) supplemented with 25 mM imidazole and eluted in purification buffer with 300 mM imidazole. The eluate was incubated with Strep-Tactin superflow plus resin (Qiagen) for 30 min, washed with 10 bed volumes of purification buffer, and eluted with purification buffer supplemented with 10 mM desthiobiotin (Sigma-Aldrich). The eluted fractions were concentrated by ultrafiltration using 30-kDa molecular weight cutoff Centricon centrifugal filter units (EMD Millipore) and applied onto a Superdex 200 10/300 GL column (GE Healthcare) for size exclusion chromatography (SI Appendix, Fig. S1B ). Purified fractions (SI Appendix, Fig. S1C ) were pooled, concentrated to 2 mg/mL, and frozen in liquid N2 for storage at −80 °C.
3
Purification of LwaCas13a Protein
4
RAF1 Pull-Down Assay Protocol
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After the cultivation, cyanobacterial cultures were pelleted by centrifugation at 3000×g for 10 min at 4 °C. Crude cell extracts were obtained by sonication in 20 mM Tris pH 8.0, 150 mM NaCl, 2 mM PMSF, and 2 mM β-mercaptoethanol followed by centrifugation at 15000×g for 15 min at 4 °C. RAF1 pull-downs were performed with the use of protein extracts from wild-type cells. For cell extracts containing 1 mg of proteins, 20 µg of N- or C-terminally Strep-tagged RAF1 was added and pre-incubated for 5 min at RT. After initial pre-incubation, 50 µl of Strep-Tactin Superflow Plus resin (Qiagen, Hilden, Germany) was added and binding was carried out with gentle rotation for 15 min at RT. Resin was washed five times with wash buffer (20 mM Tris pH 8.0, 150 mM NaCl). Bound polypeptides were eluted using wash buffer supplemented with 2.5 mM d-desthiobiotin. PAGE-separated polypeptides were excised from gels, trypsin-digested, and subjected to mass spectroscopy analysis at the Mass Spectroscopy Laboratory of Biophysics and Biochemistry Institute of the Polish Academy of Sciences (Warsaw, Poland).
5
Purification of the Escherichia coli ItaRT Complex
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Escherichia coli BL21(DE3) cells carrying pET22-strep-itaRT-his plasmid were grown to OD600 of ∼0.6 in 20 ml of LB medium containing ampicillin (50 μg/ml). Protein expression was induced with 1 mM isopropyl-β-d -thiogalactopyranoside (IPTG), followed by growth at 18°C for 16 h. Cells were harvested by centrifugation, washed with BW buffer (20 mM Tris–HCl, pH 8.0; 300 mM NaCl) and resuspended in 2 ml of BW buffer supplemented with 0.2 mM phenylmethanesulfonyl fluoride (PMSF), 1 mg/ml lysozyme, and 1 mM β-mercaptoethanol. The cells were lysed by sonication, and the debris was removed by centrifugation. The clarified cell lysate was mixed with 100 μl of Talon CellThru Co2+-chelating resin (Takara-Clontech, USA) pre-equilibrated with BW buffer and incubated with gentle rotation at 4°C for 2 h. The resin was washed with BW buffer, and the ItaRT complex was eluted with 1 ml of EB buffer (50 mM Tris–HCl, pH 8.0; 0.5 M imidazole, 300 mM NaCl). The eluted material was combined with 100 μl of Strep-Tactin Superflow Plus resin (Qiagen, USA) and incubated with gentle rotation at 4°C for 4 h. The resin was washed with BW buffer and eluted with 1 ml of BW buffer containing 2.5 mM desthiobiotin (Sigma-Aldrich, USA). Protein fractions were analyzed using Laemmli 12% SDS-PAGE. The gel was stained with EZblue protein stain (Sigma-Aldrich, USA) according to the manufacturer's protocol.
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Escherichia coli BL21(DE3) cells carrying pET22-strep-itaRT-his plasmid were grown to OD600 of ∼0.6 in 20 ml of LB medium containing ampicillin (50 μg/ml). Protein expression was induced with 1 mM isopropyl-β-
6
Purification of His-tagged and Strep-tagged Human ACE2
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The human ACE2 protein, tagged with His-tag and Strep-tag, was overproduced in Sf 9 insect cells. For purification, 107 cells were used. The ACE2 tagged protein was purified with the usage of Strep-Tactin Superflow Plus resin (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. Cells were lysed in the NP buffer (50 mM NaH2PO4 pH 8, 300 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100) containing proteases inhibitors (Pierce™ Protease Inhibitor Mini Tablets). The clear cellular lysate was incubated with the resin for 1 h with rotation. Next, the resin was washed with NP buffer and ACE2 protein was eluted with NP buffer containing 10 mM biotin hydrazide.
7
Purification of His-Tagged Fusion Protein
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The solubilized protein was incubated for 1 hour with washed and pre-equilibrated Strep-Tactin Superflow plus resin (Qiagen). The resin was then loaded into an Econo-Column Chromatography Column (Biorad) for gravity flow purification. The resin was washed 10 x with 2 volumes of wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.03 % (w/v) DDM).
Stepwise protein elution was performed in wash buffer supplemented with 5 mM desthiobiotin (IBA-Lifesciences). The eluted protein was then treated with His-tagged human rhinovirus 3C protease (HRV 3C) to cleave off the C-terminal eGFP fusion and the tag. Reverse IMAC was performed to remove the protease. The protein was then concentrated to 0.5 ml using a 50 kDa cutoff concentrator (Vivaspin, Sartorius, MWCO 50 kDa). Size-exclusion chromatography was carried out on a Superdex 200 increase 10/300 GL column (GE Healthcare) in 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.03% (w/v) DDM, 1 μM cyanopindolol. The protein was concentrated to 3 mg/ml using a 50 kDa cutoff concentrator (Vivaspin, Sartorius). The purified protein was flash frozen in liquid nitrogen and stored at -80 o C.
Stepwise protein elution was performed in wash buffer supplemented with 5 mM desthiobiotin (IBA-Lifesciences). The eluted protein was then treated with His-tagged human rhinovirus 3C protease (HRV 3C) to cleave off the C-terminal eGFP fusion and the tag. Reverse IMAC was performed to remove the protease. The protein was then concentrated to 0.5 ml using a 50 kDa cutoff concentrator (Vivaspin, Sartorius, MWCO 50 kDa). Size-exclusion chromatography was carried out on a Superdex 200 increase 10/300 GL column (GE Healthcare) in 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.03% (w/v) DDM, 1 μM cyanopindolol. The protein was concentrated to 3 mg/ml using a 50 kDa cutoff concentrator (Vivaspin, Sartorius). The purified protein was flash frozen in liquid nitrogen and stored at -80 o C.
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