As you mention you protein molecular wt: 22 and 27 Kda and PI is 4.9 and 5.8.

The difference of PI is 0.9 unit, it will easly seperate from anion exchange like Capto DEAE or Source 30 Q resin.  at pH 8.5 to 9.0 ph with 20mM tris buffer, and give gradient 0-100 % 20 CV, keep linear flow rate less than 100 cm/h, maximum 10 mg/ml of resin you have to bound.  

Here you have to refine your salt concentration Q binding may happen with less salt like 2-3 ms/cm and DEAE will happen with little more salt like 6-10 ms/cm. it depend on protein.

still not happen find out salt concentration for elution, than give salt  with pH gradient also.

it will happen, try it...

What pH buffer are you using? and how sensitive is your protein to salts?

You should be able to separate them on IEX. What I would do is load a test sample on a small column and run with a long gradient (like 100cvs) from 50mM NaCl - 1M NaCl. You should be able to pin point what salt concentration each elutes at. For better separations you can run your gradient at slower increases or flat at your protein of interest.

I would recommend to use hydrophobic chromatography (like Phenyl-Sepharose) for separation of your two proteins having close MWs and isoelectric points. You can use 1.0 M Ammonium Sulphate (adjusted to pH 7.0 - 7.5) as a loading buffer, and then decrease the concentration of AS as a step-wise gradient elution with 0.5 M, 0.2 M, 0.1 M, 0.05 M (at the same pH), and then make the final elution with 30% Ethanol in 50 mM AmBic buffer. There is a general rule (mostly empirical), that any two proteins from the same species can not have the identical or close MWs, isoelectrical points, and hydrophobic properties. Usually, one of these three parameters is very different in these two proteins. You can try to prove it again (it was shown multiple times) with your proteins. Good luck !

Hi,

In addition to the suggestions mentioned above, you could consider running IEX in chromatofocusing mode, so run a pH gradient instead of a salt gradient. You will need special buffers then though. A combination of pH and salt gradient could als be an option to try.

Good luck!

 I do agree, Hydrophobic chromatography may be a better alternative. you can try running a very slow gradient (using FPLC) changing concentration of salt slowly and using something with salt-tolerant chromotography (primary amine) where the interaction with the proteins is stronger and requires a much higher salt concentrations... of course, if you think your proteins are tolerable to that sort of salt. May be you can avoid this by having a much careful nickel purification with a long gradient of imidazole... you might end up with those proteins in different fractions. Best of luck

Are these measured or predicted PIs?

Try a KCl gradient instead of NaCl once.

Hello thank you ALL for the answers, your suggestion are really great and for the answers to yours queries, I write below:

1. The pH for elution following in affinity chromatography is 7.0

2. And my pIs are predicted by bioinformatics.

I will try the pH gradient and the salt gradient , but I will also do the hydrophobic interaction chromatography.

Thank you ALL once more.

As you mention you protein molecular wt: 22 and 27 Kda and PI is 4.9 and 5.8.

The difference of PI is 0.9 unit, it will easly seperate from anion exchange like Capto DEAE or Source 30 Q resin.  at pH 8.5 to 9.0 ph with 20mM tris buffer, and give gradient 0-100 % 20 CV, keep linear flow rate less than 100 cm/h, maximum 10 mg/ml of resin you have to bound.  

Here you have to refine your salt concentration Q binding may happen with less salt like 2-3 ms/cm and DEAE will happen with little more salt like 6-10 ms/cm. it depend on protein.

still not happen find out salt concentration for elution, than give salt  with pH gradient also.

it will happen, try it...

One technique that has not been mentioned is isoelectric focusing.  This involves setting up a pH gradient by electrophoresis in a mixture of buffer species with differing pIs.  The proteins will concentrate at their appropriate pIs.  If carried out in a gel, the proteins can be eluted from the sectioned gel.  If carried out in a sucrose density-gradient column, fractions can be collected as the column is drained.