Analysis of proteins
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1 Analysis of proteins Western blot Protein seperation (liqiuid chromatography) Mass spectrometry Assaying of protein in... Blood (e.g. viral infections, pregnancy test) Cells Tissue Urin (bladder infection) Food (toxins) 1
2 WESTERN BLOT Although gel electrophoresis is a relatively simple technique, it is not a trivial procedure. David E. Garfin, Electrophoretic Methods (even more true for blotting...) 2
3 Overview 1. Sample preparation 2. Elektrophoresis 3. Blotting 4. Detection 4.1 Blocking 4.2 Binding of primary antibody 4.3 Washing 4.4 Binding of secondary antibody 4.5 Washing 4.6 Substrate reaction 4.7 Development on film 3
4 Sample preparation Measurement of concentration Denatureing of proteins 1% SDS (Sodiumdodecylsulfat) other reducing agents (Mercaptoethanol, Dithiothreitol) Heating of Proteins (5-10 min, 95 C) Mixing sample with loading buffer (marker) Usually µg protein can be loaded/lane 4
5 Electrophoresis separates molecules according to their charge:mass ratio SDS-polyacrylamide gel electrophoresis Even coating of proteins allows even charge distribution -> larger mass = higher total charge 5
6 Blotting and detection: Highly specific enzymes and antibody assays can detect individual proteins Ddit4 b actin 1µM Dex 100nM Rapa PC NC T3-L1 Ads Immunoblot (= Western Blot) based on affinity 6
7 Liquid chromotography (LC): Separation of proteins by size: gel filtration chromatography Add mobile phase: buffer Stationary phase: 7
8 Separation of proteins by charge: ion exchange chromatography Also: Reversed-phase LC: separation by hydrophobicity Stationary phase: non-polar, Mobile phase: moderately polar 8
9 Separation of proteins by specific binding to another molecule: affinity chromatography 9
10 Mass spectrometry the analysis of complex protein mixtures Genome databases allow prediction of genes -> protein primary structure Each protein can be fragmented into peptides which are composed of amino acids (aa) Each peptide has a unique mass/charge ratio at a given ph Each protein therefore has a unique peptide-fingerprint Technique: proteins->peptides->mass/charge ratio measurement -> compare against whole proteome (genome based) database -> identify proteins 10
11 Mass spectrometry workflow Sample preparation Ionization source Mass analyzer Detector and data bank comparison HPLC... High-pressure liquid chromatography MALDI... Matrix-assisted laser desorption/ionization ESI... Electro-spray ionization 11
12 Time-of-flight mass spectrometry measures the mass of proteins and peptides Matrix-Assisted-Laser-Desorption/Ionization Time-offlight mass spectrometry (MALDI-TOF MS) 12
13 MS spectrum 13
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18 Major finding of this study: onset of diabetes could be reversed by disease monitoring (despite lack of risk factors (BMI= ; non-smoker) 18
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