Bio-Rad Profinity IMAC Resins - Manual (Page 20)

16
Method
1. Harvest E. coli from an appropriate volume of bacterial culture by
centrifugation at 4,000–8,000 x g for 5–10 min at 4°C.
2. Discard supernatant and place centrifuge bottles on ice.
Alternatively, the pellet may be stored at –70°C for later use. In this case, it is
recommended that the pellet be stored in flat containers or bags to allow for
easier thawing.
3. Determine weight of pellet.
This is easily done by subtracting the weight of an identical, empty container
from the weight of the one containing the E. coli pellet.
4. Resuspend pellet in 1:10 ratio (w/v) in lysis buffer and add Lysonase
bioprocessing reagent (20 µl/g of cell paste).
Thoroughly resuspend the pellet by vortexing.
Following lysis, the lysate often becomes very viscous due to the release of
genomic DNA into the solution. It is very important to reduce viscosity using a
homogenization method (step 5), as a viscous heterogeneous solution may
clog the column.
Optional lysis step:
Resuspend pellet in 1:10 ratio (w/v) in B-PER/5 mM DTT. After
resuspending, add Lysonase bioprocessing reagent, 20 µl/g of cell paste
and shake at 150 rpm at room temperature for 20 min. Proceed to step 6
below.
B-PER may be used when equipment required for mechanical disruption is
unavailable in the laboratory.
5. Sonicate the cell suspension/lysate 4 times at 1 min intervals each.
Sonicate on ice at all times.
Decrease interval time if the sample becomes warm. Keep samples cold at all
times. Check for clarity and increase sonication if needed.
6. Centrifuge the homogenized lysate at 12,000 x g for 20 min at 4°C to
clarify sample. Save supernatant.
Centrifugation times and speeds may need to be altered in order to remove all
cell debris. If supernatant is still not completely clear, lysate should be filtered
through a 0.45 µm filter to prevent column backpressure from occurring. The
target protein is now in the supernatant.
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