The purified NPR1–Strep3 (20 μM), TGA3–Flag–HA (48 μM) and the LS7 DNA containing a palindromic TGA-binding site (underlined in 5′-CACTATT TTACTGACGTCA TAGATGTGGCG-3′, 57.6 μM) were mixed and incubated at 4 °C for 30 min. The protein–DNA complex was cross-linked with 1.5 mM BS3 (Thermo Fisher Scientific) at room temperature for 30 min and quenched with 50 mM Tris (pH 8.0). The protein–DNA mixture was further purified using a Strep-Tactin column (Qiagen) and size-exclusion chromatography (Superose 6 increase 10/300 GL, Cytiva) in a buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM DTT and 0.2 mM SA.
Streptactin column
Manufactured by Qiagen
The StrepTactin column is a laboratory equipment used for the purification of proteins tagged with the Strep-tag II affinity tag. It is designed to efficiently capture and purify these tagged proteins from cell lysates or other complex samples. The column utilizes the high-affinity interaction between the Strep-tag II and the StrepTactin resin, allowing for the selective isolation of the target proteins.
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Lab products found in correlation
Superose 6 increase 10 300 gl, by Cytiva (1 mentions)
Ktaprime plus liquid chromatography system, by GE Healthcare (1 mentions)
Biolock biotin blocking solution, by IBA Lifesciences (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Gst trap, by GE Healthcare (1 mentions)
Heparin column, by GE Healthcare (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Benzonase, by Merck Group (1 mentions)
5 protocols using streptactin column
1
Characterization of NPR1-TGA3 Protein-DNA Complex
2
Purification of Twin-Tagged HIV Envelope Proteins
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Secreted twin StrepTagged gp16-V1V2 proteins in the harvested and filtered supernatant were supplemented with BioLock biotin blocking solution (IBA Lifesciences) at 5 µl/ml to mask the biotin present in the supernatant. After 30 min of incubation, the supernatant was loaded onto a 1 ml StrepTactin column (QIAGEN) at a flow rate of 0.7 ml/min in the ÄKTA prime-plus liquid chromatography system (GE Healthcare). Non-specifically bound proteins were washed off by passing at least 20 column volumes of the wash buffer (300 mM NaCl, 50 mM Tris-HCl, pH 8) or until the absorbance reached the baseline level. Bound gp16-V1V2 proteins were eluted with StrepTactin elution buffer (5 mM d-Desthiobiotin, 300 mM NaCl, 50 mM Tris-HCl, pH 8) at a flow rate of 1 ml/min. Eluted peak fractions were buffer exchanged into 100 mM NaCl, 50 mM Tris-HCl, pH 8 buffer. Protein fractions were stored with 10% glycerol at –80 °C until use for antigenicity and immunogenicity studies. GnTi expressed His-tagged gp140s and gp120s were purified from the harvested and clarified supernatant using Ni-NTA agarose beads (Qiagen) following manufacturer’s instructions.
3
Affinity Purification of Proteins
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Supernatants were loaded onto a StrepTactin column (QIAGEN) or GST trap (GE Healthcare) pre-equilibrated with wash buffer composed of 50 mM Tris HCl pH 8.0, 300 mM NaCl (180 mM NaCl for protein complexes), 5% glycerol and 2 mM DTT. The column was washed with 10 column volumes (CV) wash buffer before proteins were eluted with 5 CV wash buffer containing either 2.5 mM desthiobiotin (StrepTactin) or 10 mM reduced glutathione (GST). Depending on the experiment, tags were cleaved overnight at 4°C using PreScission (3C) protease and a protease to protein molar ratio of 1:50.
4
Purification of RSV L-P Polymerase Complex
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Human RSV L protein (strain A2) with an N-terminal dual StrepTag and RSV P protein (strain A2) with a C-terminal 6x His-tag were co-expressed in Sf9 insect cells using pFASTbac Dual transfer vector (Life Technologies). The Sf9 cells expressing the L–P polymerase complex were lysed by two passes through a microfluidizer in Strep Buffer A (50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, and 1 mM Tris (2-carboxyethyl) phosphine (TCEP), pH 8.0) supplemented with Complete EDTA-free Protease Inhibitor Cocktail (Roche) and 1 U/mL Benzonase (EMD Millipore). After clarification by high-speed centrifugation, the cell lysate was loaded onto a Strep-Tactin column (QIAGEN), and the bound protein was eluted using 10 mM desthiobiotin in Strep Buffer A. The eluted L–P complex was pooled and diluted with an equal volume of heparin buffer A (50 mM Tris-HCl, 10% glycerol, and 1 mM TCEP, pH 8.0), and was further purified using a heparin column (GE Healthcare). The L–P complex was eluted from the heparin column using a NaCl gradient to a final concentration of 500 mM. The protein was concentrated with an Amicon Ultra centrifugal filter, then loaded on to a size-exclusion column (Superpose 6 Increase 10/300, GE healthcare) equilibrated in 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, and 1 mM TCEP, pH 8.0. The fractions near the maximum height of the peak were combined, flash-frozen, and stored at −80°C.
5
Atg3 phosphorylation by Atg1 kinase
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Atg3 was immobilized on a StrepTactin column (QIAGEN) and phosphorylated using recombinant Atg1 (100 nM). Phosphorylation reactions were carried out in 20 mM HEPES-NaOH pH 7.4, 180 mM NaCl, 2 mM ATP, 10 mM MgCl2 for 4 hours at 30°C. The phosphorylated E2 was eluted in StrepTactin elution buffer (50 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 2 mM DTT, 2.5 mM desthiobiotin) and further purified using an anion exchange and size exclusion chromatography step (using a Resource Q and Superdex S75 column respectively). Fractions containing the phosphorylated E2 were pooled and concentrated. For direct control experiments the “non-phosphorylated” E2 was purified identically as the pre-phosphorylated E2, however, catalytically inactive Atg1 (Atg1D211A) was used instead of wild-type Atg1.
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