Bio-Rad Profinity IMAC Resins - Manual (Page 22)

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Section 8
Preparation of Clarified E. coli Lysate Using
Denaturing Conditions
This guideline describes a general method for the preparation of inclusion bodies
from E. coli. After cell lysis, inclusion bodies are separated from cell debris and then
resuspended/washed in a solution containing urea. For purification, inclusion
bodies must be solubilized in a high concentration of denaturant.
To reduce viscosity of the cell lysate, Lysonase bioprocessing reagent (rLysozyme
solution plus Benzonase nuclease) can be used to break up bacterial DNA.
Protease inhibitors, such as phenylmethane-sulfonyl fluoride (PMSF) may also be
included in the lysis buffer to prevent proteolysis from occurring.
An optional lysis step in the protocol is included when equipment for mechanical
disruption may not be available. This uses B-PER bacterial protein extraction
reagent, which may be used for both soluble and inclusion body purification of
recombinant proteins from E. coli.
Always work quickly and keep cells at 4°C at all times during this procedure.
Materials
Reagents
• Denaturing lysis buffer, pH 8.0 (urea-based)
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 5 mM imidazole
– Up to 8 M urea
– Add Lysonase (Novagen catalog #71230-4), or PMSF just prior to
use in step 4
• Optional lysis buffer
B-PER bacterial protein extraction reagent (Pierce catalog #78243), add
just prior to use in step 4
• Resuspension/wash buffer for inclusion bodies: 1x PBS, 8 M urea
Equipment
• Filter apparatus (0.45 µm)
• Sonicator
Biological Sample
• E. coli with respective recombinant protein
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