BIOL 301 Lecture 8: November 4th 2016 – Lecture 8
5 Mar 2018
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November 4th 2016 lecture 8, lab 8. Purification of proteins and/or protein complex: methods and principles. To study and/or confirm protein-protein interactions in a protein complex. To assess their biochemical function in vitro (e. g. enzymatic assay) To determine their 3d-structure (e. g. x-ray crystallography) Steps needed to purify a protein of interest from cells/tissues. Gentle lysis methods: (want to keep structures intact: 1. Osmotic lysis (cultured animal cells: suspend cells in hypo-osmotic solution, 2. Enzymatic lysis of cell walls (plant, bacteria, yeast: lysozyme (bacteria), cellulase and pectinase (plant), lyticase (yeast) Glass beads (cell suspension, yeast: using abrasive vortexed bead to break cell walls. Pellet will be unbroken cells: slow speed (low g force) centrifugation to remove unbroken cells. Even simpler: property: binding to a specific molecule by affinity chrom. Enriching protein extract by salting it out: precipitation with. Separation by size and shape: gel filtration chromatography.
