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Amylose Affinity Chromatography of MBP 179
3. Methods
3.1. Amylose Affinity Chromatography at Small Scales Using
Magnetic Beads
1. Pre-cool 2.5 mL of the wash and elution buffer on ice for 20 min.
2. Wash 100 μL of amylose magnetic bead suspension by adding to 400 μL of
column preparation solution (see Note 10) in a microfuge tube and thoroughly
vortex.
3. Pull beads to the side of the tube using a magnet (30–60 s) and decant the
supernatant with a pipette.
4. Repeat step 2 by adding 500 μL of ice-cold column buffer and repeat step 3.
5. Carefully add 500 μL of clarified bacterial cell lysate (see Note 11) to
the magnetic beads and gently mix to a suspension with the pipette slowly
(see Note 12).
6. Incubate the suspension for 45 min at 4°C on a suitable shaker (e.g., rocking or
platform) at a low speed setting (see Note 13).
7. Apply the magnet and decant supernatant as in step 3. Retain the supernatant as
required in a separate microfuge tube for analysis of the unbound flow-through.
Repeat this washing step three times.
8. The functionalized magnetic beads can now be used in a secondary capture
system employing the passenger species as required.
9. If desired, elute the MBP-passenger protein complex from the magnetic beads
by adding 50 μL of elution buffer (see Note 14) and resuspend gently with a
pipette and incubate for 10 min on ice.
10. Resuspend gently with a pipette and apply the magnet as in step 3 retaining the
supernatant containing the MBP-passenger protein.
3.2. Calculation Experiment
1. Collect a 1 mL aliquot of cells at the time of harvesting the expression culture
(see Note 15).
2. Pre-cool equilibration, wash and elution buffers on ice for 20 min.
3. Lyse bacterial cells (see Note 11) and prepare a cleared lysate in 500 μL of wash
buffer 1.
4. Place supernatant in a fresh microfuge tube.
5. Wash 0.1 mL of amylose agarose or amylose high flow suspension by adding
to 0.9 mL of column preparation solution in a microfuge tube and thoroughly
vortex.
6. Pellet the matrix by centrifugation at 2000 × g for 1 min and decant the supernatant
(see Note 16).
7. Wash once with 1 mL of pre-equilibration buffer, thoroughly vortex and repeat
step 6.
8. Resuspend in 0.5 mL of equilibration buffer and transfer to a fresh microfuge
tube (see Note 17).