selection of column depends on protein properties. in general we don't prefer cation exchange chromatography. in your case i have no idea about your protein properties, but you are using week cation exchange chromatography, so there may be chance of extra bands on your gel. better you use strong anion resin like like Q separose, QAE etc for protein purification.

one impotent thing is, for ion exchange chromatography your equilibration buffer buffer buffer pH must be ±1 point above or bellow than your protein PI and your protein solution pH should match with your equilibration buffer. 

better seperate your protein by salt salt gradient. if it  don't work try isocratic gradient.

if you need more clarification contact me on : [email protected]

You should give more details about your purification, what type of purification have you made? Usually it's neccessary to perform several steps of purification in order to get a protein pure enough. But there are a lot of posssibilities depending on the protein construction and properties; affinity (nickel, cobalt, GST, MBP...), size (gel filtration), charge (ion exchange), etc. Please, give more details and we will please to help you.

I am agree with Arshad Jawed. Without any details about either approximate molecular weight or pI value one can not really suggest.

I agree with Arshad Jawed and would like to continue the list of clarifying points.

- Is your protein a soluble one or a membrane bound one?- Have you used detergent during the purification steps?- Is your protein a recombinant one (with tag) or a native one?

- Why have you chosen a weak anion exchange resin for purification? Give us reason, please, in order to help you.

The simplest way to start with ion exchange is to pick a column that you know will bind your protein based on its isoelectric point and the pH of the buffer you want to use.

Then, start with a gradient of 0-100% buffer B (buffer B should be buffer A + 1 M NaCl) and the gradient should be ~10 column volumes long. Collect every peak, run the gel, and modify the gradient from there. If you still have multiple proteins with your desired protein, try extending or changing the gradient in that region. Or try using the opposite ion exchange resin after dialyzing out the salt (i.e. SP instead of DEAE or Q). 

Hope this helps. As the others said too, if you provide more info, we can be more helpful.

You need to know first your protein PI, based on the PI you can select whether to go for anion exchange and cation exchange. Binding buffer should have lower NaCl concentration perhaps around 30-50mM and elution buffer with 500mM to 1M NaCl.

DEAE-Sephadex chromatography can purify your protein . Swell the gel for 72hrs at room temperature. Pack in column with 25 cm length and 2.5cm width. After washing the column pack it with gel load sample protein collect fractions till the protein un-adsorbed shows near zero OD. Wash the bound protein by using linear discontinuous gradient 0.1M-0.4M KCl every time till zero OD at 280nm.It should yield you sufficient purification. If DEAE-Cellulose is used it need to be activated by HCl and Naoh treatment. It is better to use DEAE-Sephadex

Hello everyone, sorry for the late response.

My protein of interest is around 60kd molecular weight and PI is around 4.2. It's a recombinant protein with His-tag in pET28a.

Its resulting in an inclusion bodies and expression is really high. I used lysozyme, triton-x 100, 25mM glycerol and 2 rounds of sonication for 10mins with 50 amplitude.

I did manage to get it in to soluble fraction but very less amount of protein.

I did go ahead to purify and elution is less with non-specific bands.

Looking forward for your suggestions.

Have you tried purify the protein from the inclusion bodies? If the expression is higher and in inclusion bodies you'll get a best quality protein.

Hello Arshad,

Thank you!

I did standardize urea of 8M and used in same way as you mentioned. But, would not it be a problem in refolding the purified protein and might affect the downstream process?

Hi Megha,

Are you doing affinity purification at first place? if you have inclusion bodies, lyse your cells in Tris pH-8, 500mM NaCl, 8M uear or 6M Gn HCl, 2mM DTT and load on affinity column or use loose beads. Once you have done, exchange the buffer based on what type of column you want to use, it is also based on the PI of your protein,

here are some suggestions

In a buffer with a pH greater than the pI of the protein of interest, the protein will carry a net negative charge; therefore, a positively charged anion exchange resin is chosen to capture this protein.

In a buffer with a pH lower than the pI of the protein of interest, the protein will carry a positive net charge; thus a negatively-charged cation exchange resin is chosen.

use about 3-50mM NaCl in binding buffer (bufferA), and 1M NaCl in elution buffer, do gradient elution,  concentration of NaCl gradually increases and elutes pure protein from ion exchange column.

Hope this helps

Thamarai

Thank you Folks for your immense suggestions.

And, the problem is fixed.