BIOL-L211 Lecture 35 Outline of Last Lecture I. ArticleII. Signal TransductionIII. Protein detectionOutline of Current Lecture I. Protein PurificationCurrent LectureProtein PurificationI. Protein PurificationA. Cell Extract: made by bursting open a cell so that it releases its proteins1. Cells burst with detergents/shearing forces/salt/etc.2. Lysate: mixture of proteins that escapes from the burst cell3. Lysate can be purified to study specific proteinsB. Column Chromatography: most common protein-purification method1. 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 the column2. In all cases, multiple fractions are collected (each containing a slightly differentset of proteins); these fractions can be assayed for additional information on the proteins3. Ion-exchange Chromatographya. Utilizes charged beads; separates proteins based on their surface chargeb. Example: Negatively charged beads will bind positively charged proteins so that they remain in the column while other proteins are eluted; flushing the column with a high concentration of salt will release the positively charged proteins for further analysisThese 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 Chromatographya. Utilizes beads with varied pore size; separates proteins by size and shapeb. Example: If beads have small pores, large proteins will elute faster while the small proteins travel through the pores and take longer to elute5. 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 columnc. Bait: the protein for which the antibodies are specificd. Prey: the protein being tested for interaction with the baite. If the prey binds the bait, it will not elutef. The antibody-protein (bait) complex can then be elutedg. SDS-PAGE and Western blot can be performed with antibodies specific to both proteinsh. Protein-protein interaction will yield two bands on the western
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