Thermal stability of the purified wild-type Vpb4Da2 and variants in protein storage buffer was performed in triplicate reactions using 25 μL of 0.5 mg/mL protein and 5 μL of 10x SYPRO™ Orange (Thermo Scientific). The CFX96™ real-time PCR detection system (Bio-Rad) was used to follow a thermal denaturation gradient of 20°C to 95°C with an increment of 0.5°C per 5 Sec. Fluorescence data were fit using the CFX manager 2.1 (Bio-Rad).
Superdex 200 10 300
Superdex 200 10/300 is a size exclusion chromatography column designed for the separation and analysis of proteins, peptides, and other biomolecules. The column has a bed volume of 24 mL and is suitable for use with ÄKTA™ chromatography systems.
Lab products found in correlation
14 protocols using superdex 200 10 300
Analytical SEC and Thermal Stability of Vpb4Da2
Thermal stability of the purified wild-type Vpb4Da2 and variants in protein storage buffer was performed in triplicate reactions using 25 μL of 0.5 mg/mL protein and 5 μL of 10x SYPRO™ Orange (Thermo Scientific). The CFX96™ real-time PCR detection system (Bio-Rad) was used to follow a thermal denaturation gradient of 20°C to 95°C with an increment of 0.5°C per 5 Sec. Fluorescence data were fit using the CFX manager 2.1 (Bio-Rad).
Purification of Recombinant Parkin Oligomers
The cell lysates were centrifuged at 48,000 g for 30 min, at 4 °C. The supernatant of GST-parkin was purified on a 20 ml GST column and eluted with 10 mM reduced glutathione. The pull-down fractions were concentrated using Amicon Ultra concentrators with 10 kDa Millipore membrane and separated by gel-filtration on Superdex200 10/300 (Cytiva, Uppsala, Sweden). Purified on affinity chromatography, parkin was eluted from size-exclusion Superdex200 column in V0 as oligomers with molecular weight not less than 1000 kDa.
Purification of BTB/POZ Proteins
The BTB/POZ domains of the potassium channel tetramerization domain proteins prevalently assume pentameric states [45] (link).
The full-length sequence of KCASH2 was cloned in pGEX4T3 using the restriction sites BamHI and EcoRI. The recombinant protein GST-KCASH2 was purified by a GST-Trap FF (Cytiva, Milan, Italy). The fraction of interest was dialyzed against 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM DTT at 4°C and successively digested with the specific protease (Thrombin, SIGMA Aldrich). Successively, the sample was loaded again on the GST-affinity chromatography and finally obtained pure on a gel-filtration chromatography (Superdex 200 10 × 300, Cytiva, Milan, Italy).
Recombinant SARS-CoV-2 Protein Production
Purification of LmCpfC Protein Complexes
LmCpfC wild‐type and variants were generated, expressed, and purified as reported previously.
Protein Characterization by SEC-MALS
Labeling Recombinant Histones for Assays
Recombinant SARS-CoV-2 RBD Protein Production
Molecular Weight Determination of Proteins
Purification of His-Tagged Proteins
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