Lane 1 is overloaded, so it's difficult to assess the purity. There is one main band, but the protein is not fully purified. It's possible that there is some aggregated protein that was not fully dissociated by the SDS-PAGE sample buffer, but it's just as likely that the IEX method simply did not achieve full purification. Try adding another chromatography step to improve the purity. Gel filtration chromatography is an obvious choice. Do you have an opportunity to use affinity chromatography, such as by including a His tag in the construct?

Yes, i have access. I AFFRIAD TO re-try Affinity chromatography and gel fimtration chromatography But the problem i faced they elute all my protein. Like what happened previously after loaded sample initially protein conc 2.4mg/ml. Post-chromatography the protein conc was left 0.2mg/ml.

The protein will normally be diluted by gel filtration chromatography. Afterwards, the chosen fractions can be concentrated by ultrafiltration.

Alternatively, start with gel filtration chromatography as the first chromatography step, then load the chosen fractions directly onto the IEX column. This avoids the need to concentrate the fractions. Using this sequence also makes it practical to begin with ammonium sulfate fractionation as the first purification step, since gel filtration chromatography also removes residual ammonium sulfate from the resuspended pellet. The sequence is:

1. ammonium sulfate fractionation

2. gel filtration chromatography

3. ion-exchange chromatography

If the protein still isn't pure enough, you can add another step, such as affinity chromatography or hydrophobic interaction chromatography (HIC) at the end. HIC is a good choice to follow IEX because you don't have to remove the salt from the IEX elution.

Thank you so much for your suggestion. I will try this.