This course is focused on instrumental methods of biophysics and molecular biology. Its main goal is to explain what problems can be solved using each of the methods and to teach how to select the simplest and the most effective approach to answer particular experimental question. Practical works will include planning of simple fluorescence or CD experiments and processing real experimental data from such experiments.
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Course: 4 lectures, 4 practical works
Lectures/Seminars: Volodymyr Shvadchak
Fluorescence principles. Fluorophores. Brightness. Solvatochromism. Protein labeling with organic dyes. Tryptophan fluorescence and other natural fluorophores. FRET and its application to study protein interactions. Fluorescence anisotropy.
Electrophoresis of proteins and oligonucleotides. DLS. FCS. CD spectroscopy to determine protein structure. IR spectroscopy.
NMR. Spin. 13C and 15N protein labeling. NMR for protein structure analysis. Solid state NMR. ESR and free radicals. X-ray. Protein crystallization.
HPLC principle, preparative and analytical applications. Types of columns. Ion exchange chromatography. Size-exclusion. Mass-spectrometry. LC-MS. ESI, MALDI and other ionization methods. Types of mass detectors. Fragmentation. LC-MS in proteomics.
Fluorescence quantum yield. Solvatochromic dyes.
Characterization of protein-membrane interaction using tryptophan fluorescence.
Solving of simple problems on solution concentrations
Practice with real data: characterization of protein-membrane interaction using CD spectroscopy.
Problems on concentration determination using UV-absorbance of proteins and on protein labeling.
Practice with real data: determination of IC50 of inhibitor based on kinetic experiment data
Processing of SDS-PAGE data to quantify fractions proteins in the mixture.
Selection of proper gradient for chromatography experiment.
Advances in Protein Molecular and Structural Biology Methods (2022) https://doi.org/10.1016/C2020-0-03170-1 (Розділи 7, 8, 10, 12, 14, 16, 20, 21)
Пояснення про відображення похибок на графіках. Geoff Cumming, Fiona Fidler, David L. Vaux, Error bars in experimental biology. JCB. (2007) https://rupress.org/jcb/article-pdf/177/1/7/1329781/jcb_200611141.pdf