I am trying to purify a protein that is expressed in NEB10 beta E.coli competent cells and has his-tag. I used Ni-NTA column however Ni-NTA did not fully purify and I had activity in the flow through as well as in the fractions that contain the wash buffer meaning that some of it is eluting before any elution buffer comes through. I got a peak at around 20% buffer B (elution buffer) but I got many bands from that peak on SDS-PAGE. I also got another peak at around 80% buffer B, while this peak is better but still not pure. I moved on to do anion exchange using HiTrap QFF column (strong anion exchange) but I got activity in every fraction (That's 80 fractions, 5 mL each). The picture shows my SDS-PAGE from Ni-NTA column, 33-59 are the fractions for my first peak. 60-80 is my second peak that I ran on anion exchange and got activity in every fraction.
My other problem is I use Amicon protein concentrators, I tried different molecular weight Cutoff (10kDa and 30kDa) but I keep having activity in the filter and the flow through . what could the problem? I don't have inclusion bodies so I don't think the protein is misfolded.
Ni-NTA column wash buffer: 50mM KPi, 10% glycerol, 1mM BME, 15mM imidazole
Ni-NTA elution buffer: 50mM KPi, 10% glycerol, 1mM BME, 500mM imidazole
Anion exchange column wash buffer: 50mM KPi, 10% glycerol, 1mM BME
Anion exchange column elution buffer: 50mM KPi, 10% glycerol, 1mM BME, 500mM NaCl
Dear Alaa Aziz ,
It sounds like you are having trouble purifying your His-tagged protein using Ni-NTA and anion exchange chromatography. There are a few potential issues that could be causing this:
It's generally recommended to try multiple purification methods and optimization of the buffers and conditions to achieve the best results. Additionally, you can try to use other purification methods like size exclusion chromatography (SEC) or hydrophobic interaction chromatography (HIC) to purify your protein.
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