Journal: Molecular Genetics & Genomic Medicine
Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation
Figure Lengend Snippet: Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.
Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).
Techniques: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction