Bio-Rad Profinity IMAC Resins - Manual (Page 19)

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Section 7
Preparation of Clarified E. coli Lysate Using
Nondenaturing Conditions
This guideline covers a general method for preparation of a clarified lysate from
bacterial cells using nondenaturing conditions. Extract purification is necessary in
order to release the target His-tagged protein from cells and separate out insoluble
material that would otherwise foul and contaminate the column. 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. In the absence of strong denaturants,
proteins may be subject to degradation during the cell harvest and lysis steps.
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 for extraction of recombinant proteins from E. coli.
Always work quickly and keep cells at 4°C at all times during this procedure.
Materials
Reagents
• Lysis buffer (pH 8.0)
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 5 mM imidazole
– 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
Equipment
• Filter apparatus
• Sonicator
Biological Sample
• E. coli with respective recombinant protein
Additional Materials
• Equipment for determining total protein concentration within the lysate
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