User:Steven Moss/Notebook/AU CHEM-570/2012/04/03

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Objective

Further purify MBP with amylose column

Procedure

  1. Create column buffer with 20 mM tris, 200 mM NaCl, and 1 mM EDTA (1.0 L made and filtered)
  2. Create elution buffer with column buffer + 10 mM Maltose (only need about 100 mL)

Regeneration of Amylose Resin Column

  1. Run 90mL of distilled water over the column at 4mL/min.
  2. Run 90mL of 0.1% SDS over the column at 4mL/min.
  3. Run 30mL of distilled water over the column at 4mL/min.
  4. Run 150mL of column buffer

Purifying MBP

  1. The filtered fractions showing protein (~10mL) were pumped over the regenerated amylose resin column at 1.0mL/min.
  2. Wash the column with 100mL of column buffer at 1.0mL/min.
  3. Elute the protein from the column with 100mL elution buffer at 1.0mL/min.