IUB BIOL-L 211 - Protein Purification (2 pages)

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Protein Purification

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Protein Purification


Protein Purification/Column Chromatography

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Indiana University, Bloomington
Biol-L 211 - Molecular Biology
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BIOL L211 Lecture 35 Outline of Last Lecture I Article II Signal Transduction III Protein detection Outline of Current Lecture I Protein Purification Current Lecture Protein Purification I Protein Purification A Cell Extract made by bursting open a cell so that it releases its proteins 1 Cells burst with detergents shearing forces salt etc 2 Lysate mixture of proteins that escapes from the burst cell 3 Lysate can be purified to study specific proteins B Column Chromatography most common protein purification method 1 Lysate is filtered through a column with small beads of a controlled nature such that certain proteins elute faster while others spend more time in column the 2 In all cases multiple fractions are collected each containing a slightly different set of proteins these fractions can be assayed for additional information on the proteins 3 Ion exchange Chromatography a Utilizes charged beads separates proteins based on their surface charge are release b Example Negatively charged beads will bind positively charged proteins so that they remain in the column while other proteins eluted flushing the column with a high concentration of salt will the positively charged proteins for further analysis These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute 4 Gel filtration Chromatography a Utilizes beads with varied pore size separates proteins by size and shape b Example If beads have small pores large proteins will elute faster while the small proteins travel through the pores and take longer to elute 5 Co immunoprecipitation Co IP a Tests whether two proteins interact bait and prey b Antibodies for the first protein are bound to the beads in the column c Bait the protein for which the antibodies are specific d Prey the protein being tested for interaction with the bait e If the prey binds the bait it will not elute f The antibody protein bait complex can then be eluted g SDS PAGE and Western blot can be performed with antibodies specific to both proteins h Protein protein interaction will yield two bands on the western blot

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