I will use ion exchange chromatography with an anion exchange column to purify chicken IgY. Prior to this I did dialysis to remove salts from previous purification steps, is it possible after this step to just change buffer system?

The pI of IgY is 5.6-7.6, and so, what buffer should I use for the chromatography? I know I want the IgY to be negative, so I would want pH in start buffer to be higher than pI?

I bought a column but it has not arrived yet. Does it come with recommendations on preferred buffer system for the chromatography or can I use any one I want? And also, what buffer should I use to elute my protein? Do you have any recommendations?

In previous steps, prior to dialysis, I used phosphate buffered saline with pH 7.

Thanks in advance


1 Answer 1


Your column manual will probably come with buffer recommendations (these manuals are also often available online). You can also search for publications that have purified IgY by anion exchange chromatography and use their procedure as a starting point.

Some generalities to keep in mind for buffer selection:

  • Buffers are most effective when the desired pH is close to their pKa. A rule of thumb is to choose a buffer with a pKa within 1 unit of the desired pH.
  • For anion exchange, the pH should be higher than the pI of the protein so that it carries an overall negative charge and will bind to the positively charged column. Another rule of thumb is to keep the pH about 1 unit above the pI. If the pH is too low, the protein will not bind. If the pH is too high, the protein will require more salt to elute, which can cause instability/precipitation.
  • For anion exchange, the buffer itself should be neutral or positively charged so it does not bind to the column. This can affect binding of the target protein both by its ionic strength and localized changes in pH.
  • Choice of pH depends on whether your column is "strong" or "weak". The charge on the functional group of a strong ion exchanger will not appreciably change over a wide range of pH whereas the charge on a weak ion exchanger is only maintained over a relatively narrow pH range.
  • This may go without saying, but make sure that your protein is stable in the buffer and pH you select. You may also want to keep in mind the buffer requirements of any downstream applications and choose one that is compatible to simplify purification.

Elution is generally done with a salt gradient, though a pH gradient can also be used. In a typical setup, you would have two solutions, both with the same buffer and pH but one with high salt (let's call it B) and one with no salt (let's call it A). The sample is applied in buffer A and then a gradient from 0 to 100% buffer B is applied to the column with fractions collected periodically. The most commonly used salt for ion exchange is sodium chloride (so the counterion for anion exchange would be chloride). Since relatively high concentrations of salt are often used for elution, in general you want to avoid counterions like sulfate that have a stronger salting-out effect.

  • 1
    $\begingroup$ You're simply the best! $\endgroup$
    – Firas
    Jun 27, 2019 at 21:39

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