What your are measuring or calculating is the overall pI. But the binding to the ion exchange ligand occurs by single residues or a small patch on the surface of the protein. If you have no defined patches that can bind to either anionic or cationic ligands, then you protein cannot bind.

But this is very rare.

What is the conductivity of your sample? is it perhaps too high?

What are you using as equilibration buffer? What are your buffering substances?

What is the protein concentration?

What resins are you using?

DEAE cellulose works in basic pH. Use Tris buffer with pH 8 or phosphate buffer with pH 7.4 as per requirements. Your protein should also be in same buffer that you would be using. Elute the protein with continuous or step gradient of NaCl salt in same buffer.

Do not use deae cellulose with phosphate as buffer. Please answer the questions made by Achim Recktenwald

Yes, cellulose DEAE or similar l shrink and swell depending on conductivity.

Agarose-based ion exchange resins are by now so cheap, I would no longer use cellulose-based ion exchangers.

Answers to questions raised by Mr. Achim are: (a) my equilibration buffer is 50 mm acetate buffer, pH 5.0. (b) my buffering substances are same as my equilibration buffer. (c) the protein content of the enzyme samples are 600, 230 and 140 micrograms/ml. (c) the resin I am using is carboxy methyl sepharose, a cation exchanger. Earlier, the enzymes did not bind to DEAE cellulose under the same conditionds of buffer and pH. However, the enzyme samples are about 12% glyosylated.

Hello Christine A. Ikpeme your buffer and protein concentrations are OK; the latter even on the low side.

But I am not surprised that the protein will not bind to the DEAE ligand at pH 5. Try pH 8 with DEAE, as Ameya Dipak Bendre proposed. Tris is a good buffer at this pH.

I deliberately chose a cation resin because I don't want to toy with the pH which may affect stability. I believe purification will still take place without the enzyme of interest binding to the resin as some other contaminating proteins with opposite charges will definitely bind and separated.

I deliberately chose a cation resin because I don't want to toy with the pH which may affect stability. I believe purification will still take place without the enzyme of interest binding to the resin as some other contaminating proteins with opposite charges will definitely bind and separated. The analytical pI of the enzyme samples I am working with are 7 and 7.5. I have trouble shot as to why cellulase samples at the above pI values won't bind to a cationic resin at pH of 5.0, the only reason I am getting is the surface obscuring effect of glycosylation.

If you use a cellulase-based ion exchanger to purify cellulases, you may be losing the enzymes during a column wash before elution, or even during the loading phase. The enzyme may bind to the ligand, but then quickly cut off the ligand from the matrix, its natural substrate.

Mr. Achim, thank you for all your suggestions. But I have read severally in literature DEAE cellulose being used successfully to purify microbial cellulases. Is it not better to have the enzyme of interest separated from other oppositely charged proteins and recovered in the flow through than to make it loose stability or activity while trying to have it bound to the resin? My understanding of ion exchange purification is about separating oppositely charged proteins.

Hello Christine A. Ikpeme , I agree wholeheartedly that it is important to work with pure enzymes and not mixtures of many different proteins and/or enzymes. It cost me a year during my PhD thesis, because the enzyme preparation, which I received from others for my work was not 99% , but less than 2-3% pure.

Does your enzyme perhaps need a cofactor, a metal ion or something similar? Something you may have to add back after purification.

What are the linear flow rates you are using during chromatography?

The protein content values you give above, are this overall protein concentrations or estimates for your enzyme?

What else is in the enzyme sample mixture, which you are loading onto the column?

You could try using 25 mM acetate buffer, to reduce the ionic strength or could you try go to pH 4.5, to give it a bit more room to your pI? Both alone or in combination may make the enzyme bind a bit stronger. Normally, 2 pH-units are sufficient, but there are always exceptions.

Do you detect your enzyme in the chromatographic fractions via an enzymatic assay or using something, like SDS-PAGE? If it is an assay, could there be something in the sample that inhibits your assay?

Thank you Mr. Achim.(1) I wouldn't know at this stage if my enzymes require a cofactor or metal ion until they are characterized accordingly. (2) The linear flowrate I am using is 30 ml/hr (the pump is set at 0.5/min). (3) The protein contents are that of the flowthroughs from the action exchanger. There are 3 enzyme samples extracted from 3 varieties of sorghum malt. Already gel filtration had earlier been carried out. This cation exchange is a second step. (4) Analytical pI of the flowthroughs indicated 7, 7 and 7.5 through 96% ethanol precipitation in various buffers of 3 to 9 pH values. They precipitated maximally at 7, 7 and 7.5, respectively. (5) My enzyme activity is detected in the flowthroughs via assay with the substrate, CMC. I intend to proceed to with the SDS PAGE.

Do you do a general protein assay, like Bradford or BCA?

When did you do the ethanol precipitation, just before the ion exchange step?

Not all proteins and esp. enzymes like ethanol precipitation; acetone is usually better. But it is important to do it as cold as possible, e.g., -20C or below. If the temperature is too high, you will denature the protein.

However, you say that you did the gel filtration before the ion exchange step.

Turn it around, do the ion exchange step first. Ion exchange has a much higher capacity than gel filtration, where you can ideally only apply