- Site directed mutagenesis and cloning: This technique was used to make mutants of SOD1 protein by PCR based mutagenesis. Cloned genes were expressed using baculovirus expression vector system in Sf21 insect cells.
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- Chromatographic and salt fractionation techniques: These techniques were used for purification of normal and mutant SOD1 proteins.
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- Quantitative Western Blot: This technique was used to quantitate SOD1 proteins in cell lines/mice tissues.
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- UV-visible spectroscopy, circular dichroism, and fluorescence: These techniques were used for measuring: (i) protein concentration, (ii) enzyme kinetics, and (iii) changes in secondary/tertiary structure of proteins.
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- Mass Spectrometry: ESI-MS studies were carried out on SOD1 proteins to check their purity and also to study post-translational modifications.
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- Differential Scanning Calorimetry (DSC): DSC was used to measure thermodynamic parameters of proteins.
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- Isothermal Titration Calorimetry (ITC): This technique was used to measure the binding affinities of metal ions for normal SOD1 and its mutants. I also measured protein-protein interaction affinities for SMAD proteins.
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Novel techniques or novel use of standard techniques |
- Native-PAGE: This technique was used to characterize the different metallated forms of SOD1.
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- "Partially denaturing PAGE": Designed a novel gel system to study the subtle conformational changes in proteins due to charge, size, and shape of the proteins.
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- Bead-binding assay: Designed a novel hydrophobic bead binding assay to distinguish mutant SOD1 proteins based on their relative hydrophobicity, that cannot be easily distinguished by other standard methods.
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