Amylose resin purification

Protein Purification Protocol for MBP tagged proteins:

 

  • Preparation:
    • Prepare Lysis Buffer: 50 mL per cells from 500mL culture (+~20mL extra for later steps)
    • Add DTT to be 2 mM
    • Add 100X Protease Inhibitor cocktail (working conc.: 1X) [in -20°C freezer] and mix immediately
    • Bring an appropriate size beaker with a stir bar to the cold room
    • Column (http://www.bio-rad.com/en-us/sku/731-1550edu-poly-prep-columns)
  • Preparation of cell extract
    • Bring cell pellet from -80°C freezer and thaw on ice (~20 min)
    • Add 10-20 ml of lysis buffer into a tube and transfer cell clumps to the beaker.
    • Add the remaining buffer (leave the extra 20mL in the cylinder)
    • Stir or pipet the cell pellet until there are no clumps left (~30 min)
    • Place the centrifuge rotor and cool it down
    • After lysis is completed, pour lysate into the centrifuge tubes.
    • Use the scale and a pipette to balance the tubes.
    • Place the tubes in the rotor and place the latter in the centrifuge.
    • Start centrifugation in the setting at <20,000 rpm for 60 min.
  • Amylose resin binding
    • Equilibrate Amylose resin (2 ml per cell pellet from 1L culture) with Wash buffer
    • Pour cell extract into a 50 ml tube with Amylose resin. If volume is more than 50 ml, split lysate and resin equally.
    • Let binding proceed for 30 min with gentle rotation.
  • Washing
    • Spin a 50 ml tube containing Amylose resin and cell extract briefly at low speed (5 min, 4°C, 3k rpm).Remove majority of supernatant.
    • Transfer Amylose-resin to empty column – use fresh buffer to rinse the tube, and pour it into a column to make sure that all resin are transferred into the empty column
    • Wash resin with 12 ml of wash buffer, 3 times

 

  • Elution

 

Bound proteins or protein complexes can be eluted by the following two ways: on column tag cleavage by protease, or elution by the buffer containing 20 mM Maltose:

 

  1. Elution by on column tag cleavage:
    • Let wash buffer drain down just above a top of the resin
    • Cap the bottom of the column
    • Add 500 μl of wash buffer into column
    • Add 8 μl of GST-3C protease per 200 ml culture into the tube, and mix with resin
    • Leave it on ice or 4 degree for over night (incubation time will be adjusted by depending on the protein). Just leave it, rotation is not necessary.
    • Let the resin sit, open the column, collect then add 3 times column volume of buffer, and collect

 

Elates contain GST-3C protease, which can be removed by passing elates through 100-200 μl of glutathione (GSH) resin, or by subsequent purification using ionic exchanger column.

 

 

  1. Elution by the elution buffer

 

  • Elution
    • Elute protein with 3 column volume of the elution buffer containing 20 mM Maltose, and collect fractions