User:Susan Schultz/Notebook/Experimental Biological Chemistry/2011/09/21
|Protein Purification||Main project page|
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Purification of MBP from proteins extracted from E.coli yesterday.
Continued to follow protocols outlined in pMal fusion and purification system manual. <http://www.neb.com/nebecomm/ManualFiles/manualE8000.pdf>
The supernatant (crude protein extract) collected yesterday was added to the column which was prepped overnight. The 50 mL of supernatant was run at 1mL/min through the column, and collected in 10mL fractions. The column was then washed with 70mL of column buffer,which was also collected in 10mL fractions. The protein was then eluted using 25mL of 10mM maltose in column buffer. 5mL fractions were collected. This process was repeated using 150mL of supernatant, 75mL of column buffer wash and 75mL of 10mM maltose in column buffer elutant.
The column wash was monitored using Bradford reagent. At the end of the wash a 20μL sample of the last fraction was added to 60μL of water and 20μL of Bradford reagent. The column wash was continued until the fractions no longer turned blue, indicating that protein was no longer being washed from the column.
0.36g maltose/100mL column buffer * 1mmol/0.34230g ≈ 1mmol maltose/100mL column buffer = 10mM maltose in column buffer
A complete lack of color change was never fully achieved with the column wash, while the blue significantly dimmed it was never completely absent.
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