Hi,

Try to apply mix-mode chromatography (aka., multimodal chromatography) using ceramic hydroxyapatite (CHT) resins for the polishing step. Alternatively and conventionally you may use gel filtration resins and size exclusion methodology. But since you say your protein has high pI and CEX is the proper one, for additional interaction that CHT (cation exchange, hydrogen bonding, metal affinity (Ca+2 dependent)) can supply may contribute to getting purer fractions. As well, keep in mind that interfering protein has closer MW to your target protein that means you should choose a longer SEC column with small particle sizes and high-resolution versions of the resins. By the way, this method would also be good for the desalting purpose and to get rid of imidazole which remains from your elution buffer at the first capturing step...

Good luck,

Emir...

Dear Soy

First off all you can try to optimize you imac by inserting a washing with an buffer containig a small amout of imidazole (e.g 20-30mM) and bind the protein on the coloumn with a buffer with 10mM of imidazole.

I think that considering that there is difference in MW beetween the protein and the impurity (that could be also degraded protein) you can try the SEC (using a supedex75 coloumn) as second step.

One other possibility is insert a TEV digestion site between the Tag and the protein and perform a second subctractive imac step after the tev digestion, but in this case you will succed only in case the contaminant in the SDS-page is an impurity and not a truncated form of your protein.

On my blog. ProteoCool you can find the following video:

- ProteoCool n°13(Subctractive IMAC)

- ProteoCool n°28 (Zinc an alternative cheap and healthy metal for IMAC)

- ProteoCool n°16 (3 Common mistakes in buffer preparation for protein purification)

that are not specifically dedicated to the solutuion of your problems but contains different hints about buffer composition and subctractive imac that can be usefull for you evaluation.

good luck

Manuele

Your protein looks "pure" after imac (only two bands, which is rare). Anyway as pointed out switch conditions in imac. Switch to cobalt ions, test initial imidazole concentrations of 5, 10, 15, and 20 mM, and increase salt concentration to 500 mM. I would not run size exclusion chromatography for such close molecular weights. In the case of cex, try increasing the adsorption pH. Finally, dilution will always occur if dispersion is important.

I would approach this problems by optimizing the IMAC.

As Manuelle correctly mentioned already adding small amounts of imidazole will increase purity levels. But you can also try to change the resin / magnetic bead that you are using.

I assume you are using a Ni-NTA products, because they are the most commonly used ones. Try Co-NTA instead. It may has less protein yield that Ni-NTA but the specificity of Cobalt to the His Tag is much better than nickel.

Ni-NTA tends to high protein yields, but impurities.

1. Can you tell me your SDS-PAGE sample preparation protocol?

2. Are there any Asp-Pro regions in the amino acid sequence of your protein?