User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2011/09/21

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Objective

The objective today is to continue to purify the maltose binding protein (MBP).

Description

  1. Run 50mL of the supernatent from yesterday over the column at 1mL/min. Collect 10mL fractions.
  2. Wash the column with 70mL of column buffer at 1mL/min. Collect 10mL fractions.
  3. Elute the column with 25mL of 10mM maltose in column buffer at 1mL/min. Collect 5mL fractions.
  4. Run 150mL of the supernatent from yesterday over the column at 1mL/min. Collect 10mL fractions.
  5. Wash with 75mL of column buffer at 1mL/min. Collect 10mL fractions.
  6. Elute the column with 75mL of 10mM maltose in column buffer at 1mL/min. Collect 5mL fractions.

Data

Notes

  • 10mM maltose(100mL) = 0.36g maltose + column buffer
  • We did both of our washes until the "Bradford-by-eye" revealed that no more protein was being washed off of the column.
  • A "Bradford-by-eye" is mixing 20μL of Bradford dye with 60μL of water and 20μL of a fraction. When the mixture is bright blue protein is present, a lighter blue means there is less protein, and when the mixture is orange little to no protein is present.



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