Biochem Review Exam

Biochem Review Exam - Biochem Review Exam #2 0. DNA...

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Biochem Review Exam #2 0. DNA Purification -ultracentrifugation in CsCl gradient + High yield of DNA + High purity and DNA can be obtained - Need expensive centrifuge - Relatively time consuming (6-24 hours) -chromatography based technology + Not as time consuming (10-60 min) + Purity is good - DNA obtained this way is not nuclease free I. DNA analysis -DNA sequencing +Ultimate Way of DNA analysis b/c you get a complete DNA sequence as a result - DNA has to be purified -Restriction Enzyme Analysis + Fast, express method - Not entire DNA sequence is analyzed -Southern Blot (related to DNA arrays) +Don’t have to have purified DNA; DNA can be analyzed in complex mixtures +High sensitivity (can detect very small amount of DNA) - Not entire DNA sequence is analyzed 5’ terminus is labeled w/ P-32 (radioactive isotope) 1. Protein Purification -protein will be degraded by proteases (which is a class of enzymes which cleave/degrade other proteins) and example is trypsin 1 st step in purification - Cell lysis/Protein Extraction -To obtain cell extract (needs to be done fast) -Often protease inhibitors are used to minimize protein degredation -Cells can be solubilized in the presence of detergents (polar head, hydrophobic tail) *Disadvantage of det: Hard to remove & may affect enzymatic activities in unpredictable ways 2 nd step in purification - Preliminary Fractionation -Removal of unbroken cells and large debris -Most often accomplished by differential centrifugation 3 rd step in purification - Multiple Chromatography Stages - typically 2-3, sometimes 4-5 - Column Chromatography (separate molecules with different properties) A. Gel Exclusion Chromatography/Gel Filtration/Gel Permeation -similar to agarose gel electrophoresis -separates proteins based on size Principle : Maintain constant flow of buffer; small molecules diffuse into beads & large molecules move around. Therefore large molecules move faster through the column than the small molecules -typically this is used as the last polishing step in protein purification
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B. Ion Exchange Chromatography -most widely used -more than 100 fold purification of a target protein in single step -separation of proteins based on their differences in charge - Isoelectric point : the pH value at which the protein is neutral Principle: Typically column matrix has a charge. Beads with positively charged surface can bind negatively charged protein at alkaline pH. –Pos charged proteins are eluted. –Neg charged proteins can be eluted by adding salt solution (proteins with increasing multiple charges are eluted gradually as [salt] inc.) C. Hydrophobic Interactions Chromatography (HiC) -separates proteins based on their differing hydrophobicity Principle: Resin particles in column have hydrophobic surfaces which attract hydrophobic surfaces of proteins. Proteins which are not hydrophobic are eluted. Hydrophobic proteins will hold more tightly in high salt concentration. As the salt concentration is lowered, hydrophobic proteins are eluted in order from
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This note was uploaded on 04/09/2011 for the course CHEM 4461 taught by Professor Max during the Spring '08 term at Lamar University.

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Biochem Review Exam - Biochem Review Exam #2 0. DNA...

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