lecture_7 - Protein Purification February 5 2003 A basic...

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Protein Purification February 5 2003
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“A basic comprehension of the methods described here is necessary for an appreciation of the significance and the limitations of the information presented in the text
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Protein Isolation Must have sensitive method for detection. Select a good source for the protein. a. Rich source of material. i.e. Heart mitochondria for cytochrome C b. baker’s yeast ( Saccharomyces cerevisiae ) c. Escherichia coli (recombinant expression) Tissue specificity: Brain vs. kidney vs. eye. Chickens, cows, pigs or rats are often used. Molecular cloning techniques have allowed biochemists to over-express desired proteins in bacteria or C.H.O. ( C hinese H amster O vary) cells by isolating the gene and placing it into a host system.
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Methods of solubilization animal cells Cells can be lysed by hypotonic shock. Cells with high salt inside and no salt outside will swell and rupture Bacteria outer membranes must be digested. Gram-negative bacteria Hen egg white lysozyme digests β (1-4) linkages in the (glycosidic bonds) of polysaccharides. Mechanical breakage blenders homogenizers French press - high pressure 20,000 lbs/in 2 forced through a small hole disrupts cells
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Centrifugation Lysate - broken (lysed) cells- can be separated using differential centrifugation RPM - “spun down” separates by density differences or by size (MW) of particles. Cellular fractionation can separate: mitochondria microsomes ribosomes soluble proteins
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Centrifugation: Units ( 29 Nf V M dt r d r v s ρ ϖ - = = = 1 ln 1 2 2 Where: ϖ = angular velocity v = velocity of particle R = distance from center of rotation M = molecular weight V = partial specific volume of particle ρ = density of solvent Sedimentation velocity (Svedberg Coefficient) S = s x 10 -13
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H-bonds, ionic bonds, Van der Waals interactions, and Hydrophobic interactions can be disrupted. Denaturation is the process by which a protein loses its “native” or active shape or conformation. Temperature can play a role “cold labile” “heat labile” Protect against-Proteases, Inhibitors, Changes in pH, Protein can be air-denatured -egg white meringue - absorption to surfaces Damaged by oxidation 0 2 Heavy and transition metals damage proteins -they bind to protein- Cu + Hg + Bacterial contamination can destroy the protein Stability: proteins can denature!!
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In order to follow the purity of an enzyme, you need a method to measure its activity . Spectraphotometric analysis- is one common method to
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lecture_7 - Protein Purification February 5 2003 A basic...

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