It's hard to give a good recommendation without knowing what you've tried already. Typical protein purification of a protein with a His tag is done with metal affinity chromatography with a Ni column. After elution from the Nickle column you can then proceed to do size exclusion chromatography if you can. Let me know what you've already tried and I'll see if I can come up with some better recommendations for your unique situation.

Hello Laxmipriya Prusty

You may use IMAC (i.e., Immobilised Metal Affinity Chromatography). This technique relies on the specific interaction between the His tag and immobilized metal ions (commonly nickel or cobalt) on a chromatography resin. The His-tagged STAT1 protein will bind to these immobilized metal ions while other proteins in the E. coli lysate will flow through the column. By washing the column with buffers containing increasing concentrations of imidazole (which competes with the His-tag for binding to the metal ions), the purified STAT1 protein can be eluted from the column.

You may use the following protocol.

1. Lyse the E. coli cells expressing the His-tagged STAT1 protein to release the cellular contents, including the target protein. Use protease inhibitors to prevent protein degradation during cell disruption. Centrifuge the lysate to remove cell debris and insoluble materials.

2. You may add the clarified lysate onto an IMAC column containing a resin charged with a metal ion (e.g., Ni-NTA). The His-tagged STAT1 binds to the metal ions on the resin. Include a low concentration of imidazole (20-40mM) in the binding buffer at this step to prevent the binding of other cellular proteins that might contain exposed histidine residues while maintaining His-tag protein binding capacity. You may also add salt (0.5M NaCl) to reduce non-specific binding.

3. The column is washed with buffer to remove unbound and weakly bound proteins. The wash buffer may also contain a higher concentration of imidazole ranging from 20-50mM. This helps remove weakly bound contaminant proteins that might have lingered after the binding step. Include NaCl in the wash buffer (e.g., 0.5M NaCl) to help reduce non-specific binding.

4. The His-tagged protein is eluted from the Ni-NTA resin by increasing the imidazole concentration, which competes with the His-tag for binding to the immobilized nickel ions. The concentration of imidazole in the elution buffer is typically much higher than in the wash buffer, commonly ranging from 150 mM to 500mM. Alternatively, you may lower the pH of the buffer (which protonates the histidine residues and weakens their affinity for the metal ions). Include 0.5M NaCl in the elution buffer as well.

5. With IMAC you can often achieve high purity. But additional purification steps such as size-exclusion chromatography or ion-exchange chromatography may be necessary to achieve higher purity for certain downstream applications.

Recipe for different buffers used in the above protocol are as follows.

Binding buffer formulation includes:

20mM Sodium Phosphate pH 7.4

0.5M NaCl

20 to 40mM Imidazole

The composition of the wash buffer includes:

20mM sodium phosphate pH 7.4.

0.5M NaCl

20-50mM Imidazole

Other additives (optional):

0.1% Triton X-100 or Tween-20

5-10% Glycerol

1mM DTT or BME

Please note: A blank run without reducing agents may be necessary prior to purification if reducing agents are included in the wash buffer.

The composition of the elution buffer includes:

20mM sodium phosphate pH 7.4

0.5M NaCl

500mM imidazole

Best,

Thanks for the reply. I already followed the steps to purify it. But I didn't get the protein. Does it go to the inclusion body? Also, in the SDS gel, I didn't get the band around 91 kDa.