User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/25

AU CHEM-570 Lab Prep

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Objective Prepare amylose resin column for purification Follow periplasmic protein purification protocol for pMAL-pIII in ER2566 Bench work Added 10mL of amylose resin to column Ran 3 column volumes of water, then 5 column volumes of column buffer (20mM TrisCl pH 7.4 with 0.2M NaCl and 1mM EDTA) To the approximately 120mL of pMAL-pIII in ER2566 (resuspended in 30 mM TrisCl pH 8.0 + 20% sucrose) was added 500μL of 0.2M EDTA Centrifuged in four separate 50mL centrifuge tubes; 10 minutes at 10,000xg and 4°C Resuspended each of the four pellets by vigorous pipetting in 25mL of 5mM MgSO4; kept the tube on ice and swirled frequently When evenly resuspended, centrifuged again for 10 minutes at 10,000xg and 4°C Poured all supernatants into one tube → this should be the contents of the periplasm, where the MBP is bound Added 2mL of column buffer to the tube Using a pump, ran the contents of the periplasm tube through the amylose resin at 1mL/min, collecting 8mL fractions in disposable glass test tubes Followed with 10 column volumes of column buffer and then 10 column volumes of elution buffer (column buffer + 10mM maltose), collecting fractions throughout; still running at 1mL/min Transformation of ligations from yesterday Standard transformation protocol, but with 20μL of DNA and a 90 second heat shock Transformed into competent DH10B Results Bradford (by eye) Cells 1A through 2F are the flow-through from the periplasm/supernatant Cells 2G through 4D are the column wash fractions Cells 4E through 6B are the elution fractions 4F and 4G were the elution fractions with highest apparent protein concentration with likelihood of being MBP