User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/02/08

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Continue expression of GFP. Continue MBP "peak 4" purification using affinity chromatography.


Expressing GFP

  1. After overnight growth, spin cells at 4500rpm at 4°C for 15 minutes.
  2. Resuspend each of the pellets in 1mL of LB broth (taken from 1L sterile LB previously made in 2800mL flasks.
  3. Add each of the resuspended pellets with broth to one of the four 2800mL flasks previously made with 1L of sterile LB.
  4. Add 1mL 100mg/mL ampicillin to each flask.
  5. Incubate these four flasks at 37°C at 165rpm until 0.6 OD is reached.
  6. Add 1mL 0.1M IPTG to each flask and decrease temperature of growth to 25°C.
  7. Let the cells continue to grow and express protein overnight.

Purifying "Peak 4" of MBP

  1. Run 40mL column buffer (20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA) at 1mL/min over the amylose resin column.
  2. Run 100mL of 10mM maltose in column buffer over the column at 1mL/min.
    • 10mM maltose(100mL) = 0.36g maltose + column buffer