I am using homemade NTA-modified hyaluronic acid particles for deGFP-His purification from E.coli based CFPS mixture. On SDS-PAGE I clearly see removal of smaller proteins (< 20 kDa) but there are still impurities left (45-75 kDa). Is there any recommendation what I can change in my washing protocol?
As wash buffer I use 20 mM Tris 250 mM NaCl 10 mM imidazole, as elution buffer 20 mM Tris 250 mM NaCl 250 mM imidazole.
10 mM imidazole seems on the low end. It depends on your bead, but with many commercial beads you can wash with 40 mM and have the his-tags still fine. If it's possible, it should reduce, if not eliminate your unwanted bands.
Add a second chromatography step to improve the purity. Choose from gel filtration, ion exchange, and hydrophobic interaction.
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