GST-moesin constructs were fused to glutathione-Sepharose beads as described previously (Bravo-Cordero et al., 2013 (link)). In brief, GST-moesin (pGEX4T1, tac promoter) was expressed in Escherichia coli and purified using glutathione-agarose beads. Talin fragments constituting the talin head domain (aa 1–433), talin rod R1–5 (aa 434–1,203), talin rod R6–10 (aa 1,205–1,971), or talin rod R11-DD (aa 1,975–2,541), R11 (aa 1,975–2,140), R12 (aa 2,141–2,291), and R13-DD (aa 2,300–2,541) were generated by PCR, excised with BamHI or SalI and NotI, and cloned into the pET30a vector (EMD Millipore). The resulting constructs were expressed in E. coli BL21, and protein expression was induced with 0.2 mM IPTG as described previously (Xing et al., 2001 (link)). Recombinant his-tagged talin protein was then purified using a HisTALON column according to the manufacturer’s instructions (Takara Bio Inc.). Protein concentration was determined and 1 µg of recombinant talin was added to 50 µl of GST-moesin beads for 2 h at 4°C, washed to remove unbound protein, and run on SDS-PAGE, as described previously (Lad et al., 2007 (link); Gingras et al., 2009 (link); Bouaouina et al., 2012 (link)).
Histalon column
Manufactured by Takara Bio
Sourced in United States
The HisTALON column is a nickel-nitrilotriacetic acid (Ni-NTA) based affinity chromatography resin designed for the purification of recombinant proteins with polyhistidine (His) tags. It provides a simple and efficient method for the capture and isolation of His-tagged proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Pet30a vector, by Merck Group (1 mentions)
In fusion cloning kit, by Takara Bio (1 mentions)
Bac to bac system, by Thermo Fisher Scientific (1 mentions)
Superdex 200 pg 16 60 column, by GE Healthcare (1 mentions)
Quikchange mutagenesis, by Agilent Technologies (1 mentions)
Pierce 660 nm protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Sw28.1 rotor, by Beckman Coulter (1 mentions)
6 protocols using histalon column
1
Purification and Interaction of Talin Domains
2
Purification of DoxA Enzyme
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At the end of the fermentation process, cells were harvested in a 50 mL tube by centrifugation (6600× g, 10 min, −4 °C) and then resuspended in 10 mL sterilized water and centrifuged (6600× g, 10 min, −4 °C) to remove supernatant. The obtained cells were finally resuspended in 5 mL lysis buffer (NaH2PO4 50 mmol/L, NaCl 300 mmol/L, imidazole 10 mmol/L, glycerol 10%, pH 8.0) and disrupted by ultrasonication at 200 W for 10 min. The cell disruption suspension was centrifuged (8000× g, 4 min, −4 °C), and the supernatant was filtered through a 0.22 μm microporous membrane to obtain crude enzyme of DoxA. The crude enzyme was further purified using a HisTALON column (Takara, San Jose, CA, USA) [64 (link)]. The protein concentration of the purified enzyme was determined by the Bradford method [65 ].
3
Expression and Purification of Anhui13 NA Ectodomain
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An expression construct containing genes encoding the Anhui13 NA ectodomain (residues 75 to 465) with an N-terminal purification tag was synthesized (GeneArt). The purification tag consisted of a hexa-His tag, a human vasodilator-stimulated phosphoprotein tetramerization domain (27 (link), 28 (link)), and a tobacco etch virus protease cleavage site. The synthesized construct was cloned into the pFB-LIC-Bse vector with an In-Fusion cloning kit (Clontech). The substitution S367N was introduced by QuikChange mutagenesis (Agilent). Recombinant baculovirus was generated using a Bac-to-Bac system according to the manufacturer's instructions (Life Technologies). Following virus amplification, large-scale protein expression was carried out with 2.5 liters of Sf9 cells. Cells were removed by centrifugation at 72 h after infection, and the protein in the supernatant was concentrated and loaded onto a HisTALON column (Clontech). Fractions containing NA were pooled and dialyzed against 25 mM Tris-HCl, pH 8.0, 150 mM NaCl. The NA was further purified by gel filtration using a Superdex 200-pg 16/60 column (GE) in 25 mM Tris-HCl, pH 8.0, 150 mM NaCl. The gel-filtered protein was concentrated and stored in 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM CaCl2, 0.01% NaN3.
4
Purification and Activation of Recombinant Gαs Protein
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Recombinant Gαs were expressed in BL21 (DE3) E. coli strain and purified by affinity chromatography on HisTALON column (Clontech, Mountain View, CA) as described previously (Lee et al., 1994 (link)). Gαs was activated by incubation with 20 μM GTPγS in the assay buffer containing 20 mM Tris-HCl pH 7.8, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol for 30 min at 30°C. The unbound GTPγS was then removed by Zeba spin desalting column (Life Technologies, Carlsbad, CA). His-tagged full-length PhLP1 as well as the N-terminal (1–75 a.a.) truncation of PhLP1 were purified from E. coli as previously described (Savage et al., 2000a (link)). The purity of the recombinant proteins was assessed by Coomassie staining following gel separation and was found to be at least 80%.
For membrane preparation, striatal tissues were homogenized in buffer containing 250 mM sucrose, 20 mM Hepes pH 8.0, 1 mM EDTA, 2 mM MgCl2, 1 mM DTT and proteinase inhibitors. The homogenate was centrifuged at 2000 g to remove nuclei, followed by centrifugation at 25,000 rpm in Beckman SW28.1 rotor for 35 min in 23/43% sucrose gradient to isolate the membrane fraction. The plasma membranes were carefully collected from the layer at the 23/43% sucrose interface. The protein concentrations in plasma membrane preparations were then determined by Pierce 660nm Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA).
For membrane preparation, striatal tissues were homogenized in buffer containing 250 mM sucrose, 20 mM Hepes pH 8.0, 1 mM EDTA, 2 mM MgCl2, 1 mM DTT and proteinase inhibitors. The homogenate was centrifuged at 2000 g to remove nuclei, followed by centrifugation at 25,000 rpm in Beckman SW28.1 rotor for 35 min in 23/43% sucrose gradient to isolate the membrane fraction. The plasma membranes were carefully collected from the layer at the 23/43% sucrose interface. The protein concentrations in plasma membrane preparations were then determined by Pierce 660nm Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA).
5
Overexpression and Purification of HKDC1 and HK1
6
Overexpression and Purification of HKDC1 and HK1
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Rosetta 2 (DE3) (EMD Millipore) cells were transformed with pReceiver-B01 plasmid containing either HKDC1 or HK1. A single colony was grown overnight in 100 mL TPM media (20 g L−1 Tryptone, 15 g L−1 Yeast extract, 8 g L−1 NaCl, 2 g L−1 Na2HPO4 1 g L−1 KH2PO4) augmented with 50 μg mL−1 ampicillin and 34 μg μL−1 chloramphenicol at 37°C. The pregrowth culture was added to 500 mL of the same media and grown to an OD of ~0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown for an additional 4 hr at room temperature.
Cultures were centrifuged at 10,000 × g for 30 m and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 0.25% Tween-20, 5% sucrose, 5% glycerol, 2 mM imidazole, and 10 mM 2-mercaptoethanol) supplemented with protease inhibitors (1 mM phenylmethanesulfonyl fluoride [PMSF], 2 μg mL−1 aprotinin, 0.5 μg mL−1 leupeptin, 0.7 μg mL−1 pepstatin A). Cells were sonicated for 5 m followed by DNase I treatment (4 U mL−1) for 15 m on ice. The lysates were clarified by centrifugation at 10,000 × g for 30 m and the supernatant passed over a HisTALON column (Clontech). The column was washed with 25 mL of wash buffer (Lysis buffer pH 7.0, 25 mM imidazole). The protein was eluted from the column in 1 mL fractions using wash buffer with 500 mM imidazole. DTT was added to each fraction to a final concentration of 1 mM.
Cultures were centrifuged at 10,000 × g for 30 m and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 0.25% Tween-20, 5% sucrose, 5% glycerol, 2 mM imidazole, and 10 mM 2-mercaptoethanol) supplemented with protease inhibitors (1 mM phenylmethanesulfonyl fluoride [PMSF], 2 μg mL−1 aprotinin, 0.5 μg mL−1 leupeptin, 0.7 μg mL−1 pepstatin A). Cells were sonicated for 5 m followed by DNase I treatment (4 U mL−1) for 15 m on ice. The lysates were clarified by centrifugation at 10,000 × g for 30 m and the supernatant passed over a HisTALON column (Clontech). The column was washed with 25 mL of wash buffer (Lysis buffer pH 7.0, 25 mM imidazole). The protein was eluted from the column in 1 mL fractions using wash buffer with 500 mM imidazole. DTT was added to each fraction to a final concentration of 1 mM.
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