My protein is a mammalian protein expressed in top10 cells in E coli. Intensity of protein band at required level is good but various unwanted bands of higher molecular weights are also visible in elute sample and I want to reduce these unwanted bands.
Its is rare that a protein can be purified to homogeneity in a single step. Try an ion exchange column after the IMAC. Are you sure that this is non-specific binding, and not higher order aggregates of your protein? Such things can sometimes remain even in the denaturing conditions of a gel
If your protein is binding well, you can try reducing pH a little (but don't go lower than 7). Reducing the concentration of imidazole might also help to find where your protein is eluting, so you can reduce non-specific binding. And as always, you could use an ion exchange or gel filtration step after the IMAC. Just make sure that the protein is stable in whatever conditions you use
Increase NaCl concentration, use at least 0.5 M but preferably 1 M. Also make sure you have enough high imidazole concentration in the binding buffer. In my understanding, it is more important to control the imidazole concentration in the binding buffer than in washing buffer. Once the impurity is bound, it is tough to remove from the column, and may elute when you elute your target protein. So I would try 5-20 mM imidazole in the binding buffer and see if it helps.
Sometimes native conditions are difficult, and the only option is to go denaturing IMAC.
Hi everybody, just some more ideas.
If you are using batch purification, wash the beads with 40 mM imidazole or so before the elution and then use step-wise elution (20-40 mM) to separate unwanted impurities properly.
But the best way to achieve high purify by IMAC is to use a column and continuos purification (FPLC) - of course, the elution gradient needs to be optimized but it is worth the time. This way you can get very pure protein in one step.
You can also use some detergent in your buffer but it usually absorbs at the detection wavelength...
Playing with pH can be dangerous for your protein so be cautious.
Paul is right of course,
TOP10 cells are designed for plasmid propagation not for protein expression.
So using BL21 (DE3) - T7 expression system and something like pETxx plasmids may help your expression.
Do not bother your brain with this if the protein is abundant and not digested...
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