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- Wash the column with several volumes of binding buffer.
- Wash the column with washing buffer for native conditions
gradually increasing concentration of imidazole from 50 to
100 mM. Effi ciency of washing can be estimated by measuring
total protein concentration in washing fractions using Bradford
reagent. - Load one volume of elution buffer and incubate it on the
column for 5 min. - Collect the elution fraction and repeat elution several times
until there is no more protein detectable with Bradford
reagent. - Dialyze the protein against dialysis buffer ( see Note 3 ).
Fractions can be stored at 4 °C for several days. For longer
storage samples can be kept at −20 °C in the presence of 50 %
glycerol and 0.02 % NaN 3 or frozen as 30 μL drops in liquid
nitrogen and stored at −70 °C. Avoid repeated freezing–
thawing cycles, as it affects protein activity. - Measure protein concentration using Bradford reagent. For
better accuracy it is important to check compatibility of the
Bradford kit with high concentration of DTT prior to the test.
If needed the samples can be diluted ten times to decrease
DTT concentration. - Resuspend the cells in the lysis buffer and incubate for 1 h at
room temperature gently shaking. - Pellet down cell debris by centrifugation at 17,000 × g at room
temperature for 15 min. - Load the cleared lysate on a Ni-NTA column and let it pass
through the column several times. Incubating for 1 h at room
temperature with Ni-NTA resin can additionally increase the
yield. - Wash the column with several volumes of the lysis buffer.
- Wash the column with washing buffers for denaturing condi-
tions gradually decreasing pH from 8.0 to 6.0. Effi ciency of
washing can be estimated by monitoring total protein concen-
tration in washing fractions with Bradford reagent. - Load one volume of elution buffer and incubate it on the
column for 5 min. - Collect the elution fraction and repeat elution several times
until there is no more protein detectable with Bradford.
Elution fractions can be stored at 4 °C for several weeks or at
−20 °C for a longer period of time. - If required protein can be partially renatured by gradual dialy-
sis against dialysis buffer ( see Notes 4 and 5 ).
3.3 Purifi cation
Under Denaturing
Conditions
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