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I am confused about relationship between isoelectric point and pH in context of ion exchange protein purification. Why we cannot use this method for protein with pI below 7? Thank you very much for any tips.

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2 Answers 2

Have a look at this table, it really depends on the buffers you use:

enter image description here

The table is from this website, which is definitely worth reading when you are interested (or work) with ion exchange chromatography.

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The pI (iso-electric point) is the pH at which the protein (or other molecule), overall has a net zero charge.

As @Chris points out, the buffer you are using will change the pH the protein finds itself in. This will change the charge of the protein.

In ion exchange chromatography, there are charged chemical groups on the resin and these can cause a protein to be retained on the resin if the protein is complementary (opposite charged) to the resin.

So if you have a protein with a pI of 5 say, it would neutral with the presence of excess H+. This implies that the protein has an overall negative charge. A cationic resin would tend to retain this protein in a pH 7 buffer.

I hope that makes sense.

In most cases, ion exchange chromatography is used when most of the protein is separated out and there are a few proteins (maybe a couple of hundred) mixed together. It can get tricky if you see that the protein is mixed in with something that is also negatively charged. In that case you might change the pH of the buffer so that other negatively charged proteins are closer to their isoelectric point and come through the column faster than the one you desire to purify.

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