How to Purify His-Tagged Proteins using IMAC Method

What’s the best way to purify my His tagged protein? Supernatant super viscous after first sonication?

To purify your His tagged protein and overcome the issue of supernatant viscosity after sonication, you can follow a series of steps known as immobilized metal affinity chromatography (IMAC). Here’s a detailed guide on how to proceed:

1. Start by preparing a high-quality bacterial cell lysate expressing your His tagged protein. Ensure that you have properly induced protein expression and that the cells have been harvested and lysed efficiently

2. Centrifuge the lysate at a high speed (typically 12,000-15,000 rpm) to pellet the cell debris, as this can contribute to the high viscosity of the supernatant. Transfer the cleared supernatant into a fresh tube

3. Equilibrate a Ni-NTA (nickel-nitrilotriacetic acid) affinity resin with equilibration buffer (usually containing Tris-HCl, NaCl, and imidazole) according to the manufacturer’s instructions. Ni-NTA resin has a high affinity for His tagged proteins

4. Add the equilibrated resin to the cleared supernatant and gently mix the sample with the resin. Allow the mixture to bind the His tagged protein, ensuring good contact between the protein and the resin. Incubation times vary from 30 minutes to a few hours, depending on the specific protein and conditions

5. After the incubation, load the mixture onto a column and apply a gentle flow of equilibration buffer to wash away unbound contaminants. You may experience slower flow due to the high viscosity, but the columns are designed to handle such situations

6. Increase the stringency of the washing process by using wash buffer with a higher concentration of imidazole. This will help remove nonspecifically bound proteins or contaminants that may still be present

7. Elute the His tagged protein by using elution buffer containing a higher concentration of imidazole (e. g. , 250 mM). Collect fractions that contain your protein of interest and check their purity using SDS-PAGE or western blotting

8. After elution, you may consider using a buffer exchange technique (like diafiltration or dialysis) to remove imidazole or other unwanted buffer components that can interfere with downstream applications

During the purification process, it is essential to monitor the viscosity of the sample. If viscosity remains high after the initial sonication step, you may consider additional sonication or use a higher-intensity sonication method to further disrupt any remaining cell debris. Be cautious not to generate excessive heat during sonication to avoid protein denaturation

Remember to optimize each step of the purification process, including the choice of buffers, pH, and concentrations, to ensure the best results for your specific His tagged protein

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