153L lab 4 and 5 - Jeong Keun Kim 304346888 Chem 153L Group...

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Jeong Keun Kim 304346888 Chem 153L Group 2 Lab 4/5 Report Purification of an Alcohol Dehydrogenase from E. Coli Cells/ Characterization of Purified YqhD Alcohol Dehydrogenase- Determination of Purity, Subunit, and Native Molecular Weight Introduction In these labs, alcohol dehydrogenase “YqhD” was extracted from E. coli, purified, and was analyzed using various different methods to find the concentration, molecular weight, and the size of the protein. The E. coli were fused with the plasmid pETDuet::yqhD. This caused the cells to over-express the alcohol dehydrogenase YqhD with a His-tag attached to it. This over expressed protein was then harvested from the cell and was purified by using an affinity spin column that contained silica beads with nickel ions attached to them (Ni-NTA columns). A Bradford assay was used with a standard to find the concentration of the extracted protein. To assess the purity and the subunit molecular weight of the YqhD, the purified sample was put through a SDS-PAGE. To determine the native molecular weight of YqhD, a size exclusion chromatography could be performed. However due to the lack of sufficient equipment, this was not done in the lab. Results Standard curve
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0 50 100 150 200 250 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 f(x) = 0x + 0.03 R² = 0.99 Standard curve of known concentration [BSA] (μg/mL) Absorbance (Au) Absorbance at different purification steps Crude lysate Flow through Wash 1 Wash 2 Wash 3 Eluate 1 Eluate 2 1:10 dilution .0052 .4355 .1531 .027 -.0059 .0536 .0209 1:5 .2248 .6800 .2419 .0477 .0035 .1728 .0242 Tube [BSA] (μg/mL ) Absorbanc e (Au) 1 0 0.0991 2 25 0.1003 3 50 0.2439 4 75 0.3435 5 100 0.4335 6 150 0.5789 7 200 0.776
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dilution
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