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Lecture 13 - Proteomics

Lecture 13 - Proteomics - Proteomics the vast majority of...

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Proteomics - the vast majority of identified cells still have an unclear function overexpression of a gene gives information, but also artefacts – best method is interruption gene removal by forward genetic analysis, reverse genetic analysis (RNAi) disruption construct: sequence homology to the two flanking regions of target (20-40bp) 2D-electrophoresis 1. separation by charge (pH) – isoelectric focussing 2. first gel added to top of second 3. separation by size – SDS electrophoresis Mass spectrometry and protein identification - protein digested by trypsin proteolysis - undergoes Liquid chromatography, or more commonly, mass spec - spectral data correspond to peptide mass fingerprint, and will be unique to polypeptide - a genome database analysis will indicate what protein it is Transcription factors are modular: can be separated into DNA binding domain and the Transcriptional activating domain Yeast two hybrid method
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