Instrumental methods

General description

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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Details of the curriculum

Course: 4 lectures, 4 practical works

Lectures/Seminars: Volodymyr Shvadchak

Lectures
Fluorescence-based methods in biology

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.

Methods to determine size of the molecules and low-resolution structural methods

Electrophoresis of proteins and oligonucleotides. DLS. FCS. CD spectroscopy to determine protein structure. IR spectroscopy.

High-resolution structural methods

NMR. Spin. 13C and 15N protein labeling. NMR for protein structure analysis. Solid state NMR. ESR and free radicals. X-ray. Protein crystallization.

Chromatography and mass-spectrometry

 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.

Seminars
1. Fluorescence

Fluorescence quantum yield. Solvatochromic dyes.
Characterization of protein-membrane interaction using tryptophan fluorescence.

Solving of simple problems on solution concentrations

2. CD spectroscopy

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.

3. Basics of HTS

Practice with real data: determination of IC50 of inhibitor based on kinetic experiment data

4. Electrophoresis, chromatography and MS

Processing of SDS-PAGE data to quantify fractions proteins in the mixture.

Selection of proper gradient for chromatography experiment.

Literature
Fluorescence textbooks
Підручники загального плану
Додаткові матеріали

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)

Робота з даними

 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

Level
Undergraduate students
Lectures
4 Lectures
Practical classes
4 Lesson
Duration
2 Months
Language
Ukrainian
Lecturers

Employee of the Precarpathian University (Ivano-Frankivsk) and the Institute of Organic and Bioorganic Chemistry (Prague). Doctorate degree in Life sciences (PhD) was obtained from Strasbourg University in 2009 for research work on the development of solvatochromic fluorescent labels for studies of protein interactions.

Предмети курсу

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