BMB 471 Unit E Day 3 prelab writeup

BMB 471 Unit E Day 3 prelab writeup - Day 3...

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Day 3 - Large-scale (NH4)2SO4 precipitation • Plot of AAT/prot. vs. % (NH4)2SO4 (Results from Day 2) • Fractionation procedure with calculated (NH4)2SO4 cutoffs • Flow chart showing % (NH4)2SO4 used • Dialysis procedure • Spin column procedure • Amounts of reagents needed for all steps • Calc. for starting A340 in AAT assay BEFORE THE NEXT LAB: Using the AAT activity assays and A280 for the total protein, YOU WILL GRAPH THE DATA AND CHOOSE: 1) a lower % of sat. AS to precipitate proteins other than AAT 2) a upper % of sat. AS to precipitate most of the AAT 4 (In Day 2 Results) -Flow chart showing %AmSO 4 used (include volume and mass of AmSO 4 for each step) -Fractionation procedure (include volume and mass of AmSO 4 for each step) -Dialysis, spin column, and TCA precipitation procs. -Amounts of reagents needed -AAT assay procedure & Table A. Fractionation procedure 1. Obtain from the cold room your remaining FrII and your 100% (amm. sulfate) saturated buffer. Resuspend the solid ammonium sulfate in your buffer by stirring and place in ice while the solids settle. 2. Place a stir bar in your FrII solution. Place the beaker containing the FrII solution in a larger beaker containing ice and set both on the stir plate for continued stirring. 3. Using graduated cylinders, slowly add 115.6 mL cold, saturated amm. sulf buffer to the beaker containing FrII to make an FrII solution that is 64% saturated with ammonium sulfate. Allow to stir for 20 minutes. 4. After 20 minutes of stirring on ice, divide the saturated FrII solution into four centrifuge tubes and balance. Centrifuge at 12,000g for 15 minutes. 5.
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This note was uploaded on 03/19/2008 for the course BMB 471 taught by Professor Bowlby during the Spring '08 term at Michigan State University.

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BMB 471 Unit E Day 3 prelab writeup - Day 3...

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