hOAT variants were expressed in E. coli and the cell lysate was treated as previously described (Montioli et al., 2017 (link)). The soluble fraction was loaded on a DEAE Sepharose 26/20 equilibrated with 20 mM sodium phosphate buffer, pH 7.6. A linear gradient from 20 to 180 mM sodium phosphate buffer, pH 7.6, was then applied. Under these conditions, OAT wild-type and all the pathogenic variants elute from the column between 110 and 160 mM sodium phosphate. Active fractions were then concentrated using an Amicon Ultra 15 unit (Millipore) and applied to a Superdex 200 XK 16/60 column (GE Healthcare) equilibrated in 50 mM HEPES, pH 7.4, NaCl 200 mM. Purified protein was concentrated and stored at −20°C. The purity of each preparation assessed by SDS-PAGE was >95%. The final yield of OAT WT, Q90E, R154L, G237D, R271K, E318K and C394Y variants were 35, 4, 26, 24, 7, 11 and 8 mg/L of liquid culture, respectively.
Superdex 200 xk 16 60 column
Manufactured by GE Healthcare
Sourced in United States
The Superdex 200 XK 16/60 column is a size exclusion chromatography column suitable for the separation and purification of proteins, peptides, and other biomolecules. It has a bed volume of 120 ml and an inner diameter of 16 mm. The column is made of borosilicate glass and can withstand a maximum pressure of 5 MPa.
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3 protocols using superdex 200 xk 16 60 column
1
Expression and Purification of hOAT Variants
2
Purification of hOAT Recombinant Variants
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hOAT recombinant variants were purified from E. coli expression and subsequent cell lysis following the steps previously described [5 (link)]. The soluble fraction of the lysate was loaded on a DEAE Sepharose 26/20 equilibrated with 20 mM sodium phosphate buffer, pH 7.6. Then, a gradient from 20 to 200 mM sodium phosphate buffer, pH 7.6, was applied. Under these conditions, both hOAT wild-type and pathogenic variants eluted at a concentration of sodium phosphate between 110 and 160 mM. By using an Amicon Ultra 15 unit (Merck & Co, Rahway, NJ, USA), fractions containing the hOAT enzymes were concentrated and then loaded on a Superdex 200XK 16/60 column (GE Healthcare, Chicago, IL, USA) equilibrated in 50 mM HEPES pH 8.0, 200 mM NaCl. Purified proteins were finally concentrated and stored at −20 °C. The purity of each preparation assessed by SDS-PAGE was >95%.
3
Expression and Purification of PPM1H Protein
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HEK293 cells were cultured in Dulbecco's modified Eagle medium (Gibco) supplemented with 10% (v/v) foetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO2. Cells used in this study were regularly tested for mycoplasma contamination.
Transient transfections were performed 24 h prior to cell lysis using polyethylenimine PEIMax (0.1% w/v) (Polysciences) [39] . Cells were grown to ~70% confluency in 6-well (3. (v/v) glycerol). The resin was transferred onto a 5 ml polyprep (Biorad) filtration device.
Protein was eluted with elution buffer (wash buffer diluted with 1 M imidazole to 0.4 M imidazole) and 1 ml fractions were collected. Protein-containing fractions were pooled and 1.6 ml protein was subjected to size exclusion chromatography using a Superdex 200 XK16/60 column (GE Healthcare) and equilibrated into 50 mM Tris-HCL pH 7.5, 200 mM NaCl, 2 mM MgCl 2 , 7 mM 2-mercaptoethanol, 0.015% (w/v) Brij35. Fractions containing PPM1H were pooled and concentrated before aliquoted and snap frozen to store at -80°C. A detailed protocols.io protocol for expression and purification of PPM1H has been reported (dx.doi.org/10.17504/protocols.io.bu7wnzpe)
Transient transfections were performed 24 h prior to cell lysis using polyethylenimine PEIMax (0.1% w/v) (Polysciences) [39] . Cells were grown to ~70% confluency in 6-well (3. (v/v) glycerol). The resin was transferred onto a 5 ml polyprep (Biorad) filtration device.
Protein was eluted with elution buffer (wash buffer diluted with 1 M imidazole to 0.4 M imidazole) and 1 ml fractions were collected. Protein-containing fractions were pooled and 1.6 ml protein was subjected to size exclusion chromatography using a Superdex 200 XK16/60 column (GE Healthcare) and equilibrated into 50 mM Tris-HCL pH 7.5, 200 mM NaCl, 2 mM MgCl 2 , 7 mM 2-mercaptoethanol, 0.015% (w/v) Brij35. Fractions containing PPM1H were pooled and concentrated before aliquoted and snap frozen to store at -80°C. A detailed protocols.io protocol for expression and purification of PPM1H has been reported (dx.doi.org/10.17504/protocols.io.bu7wnzpe)
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