Rosetta2 cells were transformed with WT or p.Arg808His encoding construct of PTPN4. Overnight starter cultures were prepared by inoculating 100 mL of Luria-Bertani (LB) media (Thermo Fisher Scientific) with several colonies and grown overnight at 37°C. Large-scale cultures were grown in 2× TY media at 37°C until optical density (OD) reached ~0.6 and then induced with 0.25 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown at 18°C overnight. The cells were harvested by centrifugation and lysed in buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl, 10% glycerol, 0.25 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP,Sigma-Aldrich), 5 mM MgCl2, and protease inhibitor cocktail. PTPN4s were purified using Roche cOmplete Ni-NTA resin using manufacturer’s instructions. Proteins were further purified using Superdex 75 (GE Healthcare) size exclusion column in the final formulation buffer (50 mM HEPES [pH 7.5], 10% glycerol, 500 mM NaCl, 0.25 mM TCEP).
Complete ni nta resin
Manufactured by Roche
COmplete Ni-NTA resin is an agarose-based affinity chromatography resin designed for the purification of His-tagged proteins. It utilizes the high-affinity interaction between the Ni-NTA (nickel-nitrilotriacetic acid) ligand and the histidine tag on the target protein to enable efficient and selective purification.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 75, by GE Healthcare (5 mentions)
Luria bertani media, by Thermo Fisher Scientific (2 mentions)
Terrific broth media, by Merck Group (2 mentions)
Emulsiflex c 5 apparatus, by Avestin (1 mentions)
Isopropyl 1 thio β d galactopyranoside, by GoldBio (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
6 protocols using complete ni nta resin
1
Purification of PTPN4 Variants from Rosetta2 Cells
2
Purification of PTPN4 Variants from Rosetta2 Cells
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Rosetta2 cells were transformed with WT or p.Arg808His encoding construct of PTPN4. Overnight starter cultures were prepared by inoculating 100 mL of Luria-Bertani (LB) media (Thermo Fisher Scientific) with several colonies and grown overnight at 37°C. Large-scale cultures were grown in 2× TY media at 37°C until optical density (OD) reached ~0.6 and then induced with 0.25 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown at 18°C overnight. The cells were harvested by centrifugation and lysed in buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl, 10% glycerol, 0.25 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP,Sigma-Aldrich), 5 mM MgCl2, and protease inhibitor cocktail. PTPN4s were purified using Roche cOmplete Ni-NTA resin using manufacturer’s instructions. Proteins were further purified using Superdex 75 (GE Healthcare) size exclusion column in the final formulation buffer (50 mM HEPES [pH 7.5], 10% glycerol, 500 mM NaCl, 0.25 mM TCEP).
3
Actin Cytoskeleton Reconstitution Assay
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Previous to functionalization, an aliquot of cOmplete Ni-NTA resin (Roche) was washed three times with water in a clean disposable column. Washed beads were stored in water for up to one week at 4 °C. For functionalization, beads were carefully mixed with a total amount of 10 nmol of either formin, VCA or an equimolar mixture of both per mL of bedded resin. The binding reaction was carried out for 10 minutes on ice and stopped by washing with a 100× excess of water. Protein mixtures for network polymerization experiments were produced by mixing 12 µM profilin, 100 nM capping protein, varying concentrations of cofilin and 1 µM of CAP1 in polymerization buffer (10 mM imidazole, 3 mM MgCl2, 0,2 mM CaCl2, 1 mM DTT, 1 mM ATP, pH 7.2). The polymerization buffer was designed for slow spontaneous polymerization kinetics, therefore no additional salt was added. For experiments with VCA functionalized beads, the mixture was further supplemented with 300 nM Arp2/3 complex and the concentration of capping protein was reduced by a factor of 10 to generate clearly detectable Arp2/3 clusters. The reactions were started by first supplying the mixtures with 0,5% v/v bedded resin of functionalized beads, immediately followed by addition of 5 µM G-actin with a fraction of 10% labeled monomers.
4
Purification of Rab, Mst3, and Myosin Va Proteins
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His6 Rab10-Q63L (1–181), His6 Mst3, His6 Myosin Va GTD (1,464–1,855), and His6 Rab8A Q63L were purified in E. coli BL21 (DE3 pLys) as described by Berndsen et al (2019) (link) and Waschbüsch et al (2020) (link). In brief, bacterial cells were grown at 37°C in Lucia Broth medium and induced at A600 nm = 0.6–0.7 by the addition of 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (Gold Biotechnology) and harvested after 18 h at 18°C. The cell pellets were resuspended in ice cold lysis buffer (50 mM Tris, pH 8.0, 10% [vol/vol] glycerol, 250 mM NaCl, 10 mM Imidazole, 5 mM MgCl2, 0.5 mM DTT, 20 μM GTP, and EDTA-free protease inhibitor cocktail [Roche]), lysed by one passage through an Emulsiflex-C5 apparatus (Avestin) at 10,000 lbs/in2, and centrifuged at 13,000 rpm for 25 min in a FiberLite F15 rotor (Thermo Fisher Scientific). Clarified lysate was incubated with cOmplete Ni-NTA resin (Roche) for 2 h at 4°C. Resin was washed three times with five column volumes lysis buffer and eluted in 500 mM imidazole-containing lysis buffer. The eluate was buffer exchanged and further purified by gel filtration on Superdex-75 (GE Healthcare) with 50 mM Hepes, pH 7.5, 5% (vol/vol) glycerol, 150 mM NaCl, 5 mM MgCl2, 0.1 mM tris(2-carboxyethyl)phosphine (TCEP), and 20 μM GTP.
5
PTPN4 Protein Purification from Arctic Express
6
PTPN4 Protein Purification from Arctic Express
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Arctic Express cells were transformed with p.Arg808His encoding construct of PTPN4. Overnight starter culture was prepared by inoculating 100 mL of LB media with several colonies and grown O/N at 37°C. Large-scale culture was grown in Terrific Broth (TB) media (Sigma-Aldrich) at 37°C until OD reached ~1, then induced with 0.25 mM IPTG and grown at 12°C for 24 h. The cells were harvested by centrifugation and lysed in buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl, 10% glycerol, 0.25 mM TCEP, 5 mM MgCl2, and benzamidine protease inhibitor cocktail. PTPN4 was purified using Roche cOmplete NiNTA resin, employing a 2 M urea wash to remove excess chaperonin protein. Protein was further purified using Superdex 75 (GE Healthcare) size exclusion column in the final formulation buffer (50 mM HEPES [pH 7.5], 10% glycerol, 500 mM NaCl, 0.25 mM TCEP).
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