This procedure uses a HisTrap™ FF 1 mL column but can also be used with a HisTrap™ HP 1 mL or a HisTrap™ FF crude 1 mL column. The procedure uses ÄKTAprime plus but can also be run on other ÄKTA systems.
Alternative binding buffers: 5 to 40 mM imidazole can be included in the binding buffer to reduce unwanted binding of non-histidine-tagged proteins. The concentration of imidazole is protein dependent, and if the protein of interest elutes or does not bind at a certain imidazole concentration, reduce the concentration.
At this stage the pellet material can be washed once in buffer lacking urea and stored frozen for later processing.
The optimal concentration of 2-mercaptoethanol (0 to 20 mM) must be determined experimentally for each individual protein.
If it has not been prepared as above, adjust the sample to the composition of binding buffer by diluting in binding buffer or by buffer exchange using HiTrap Desalting or HiPrep 26/10 Desalting, then pass the sample through a 0.45 µm ﬁlter.
The requirement for linear gradient formation for refolding and elution makes the use of a chromatography system essential.
This example uses ÄKTAprime plus. Once the system is prepared, the remaining steps will be performed automatically. A model chromatogram is shown in Figure 1.
Note: If a Superloop is needed, additional information is supplied in the instructions for Superloop.
Figure 1.Results for on-column refolding of a histidine-tagged protein. For conditions, see the protocol above. Refolding of histidine-tagged membrane proteins from inclusion bodies using IMAC has also been reported.
1The columns are prepacked with Ni Sepharose 6 Fast Flow (FF) or Ni Sepharose High Performance (HP) which are precharged with Ni2+ ions.
To screen for suitable IMAC refolding conditions, a 96-well microplate prepacked with Ni Sepharose can be used (Table 2). An example is shown in Figure 2.
Figure 2. Screening of IMAC refolding conditions for histidine-tagged GFP using His MultiTrap FF. This initial screen covered buffer substances, pH and salt concentrations. Data kindly provided by J. Buchner, M. Haslbeck and T. Dashivets, Munich Technical University, Germany.
To continue reading please sign in or create an account.Don't Have An Account?