Proteins bind to the ion exchange matrices through reversible electrostatic interactions. Depending on their net charge at the working pH, the protein of interest may bind to a cation exchanger (if protein net charge is positive) or to an anion exchanger (if protein net charge is negative). Separation is achieved due to different affinities of the protein molecules to the ion exchange matrix. The bound molecules are usually eluted by increasing the ionic strength of the elution buffer (Note 1). The molecules that are weakly bound to the column elute first followed by the ones that are strongly bound. Ion exchange experiments are usually performed in the following stages:

1. 1. Preparation of ion exchanger: This includes treating the anion exchange gel material with 1 N HCl and cation exchange gel material with 1 N NaOH. This results in the ionization of all the ionizable groups present in the gel material. Repulsion between the identical charges also facilitates swelling.

2. Packing the column and equilibration of matrix: The column is packed with the binding buffer i.e. the buffer that allows binding of the sample molecules. The packed column is equilibrated with 2–3 bed volumes of the binding buffer.

3. Sample loading and binding of the molecules to column: The sample is prepared in the binding buffer and loaded on the column. This results in the binding of molecules to the counter-ionic matrix. If the sample has NaCl concentration > 50 mM , the samples should be dialyzed against the binding buffer.

4. Elution of the bound molecules: The bound molecules are desorbed and eluted either by changing the pH of the buffer or by increasing the salt concentration. A change in pH causes the molecules to reach their isoelectic points at a particular pH wherein they no longer interact with the column matrix. High salt concentration provides ions that compete with the analyte molecules for the matrix. Fractions of defined volumes are collected as soon as elution step starts.

5. Analysis of the collected fractions: The collected fractions are analyzed using the suitable methods. Protein fractions can be analyzed by recording absorbance at 280 nm or by any other protein estimation method.

6. Removal of the compounds that do not elute by the elution buffer: Some compounds may bind very tightly to the column; they can be removed by washing with very high concentration of the salt

7. Regeneration of the column: The column is regenerated by washing with 1 N HCl or 1 N NaOH.

In this experiment we are going to perform the ion exchange chromatographic experiment with hen egg white lysozyme. Hen egg white lysozyme has an isoelectric point of ~11.3 i.e. it is a cationic protein at neutral pH. We shall therefore be performing cation exchange chromatography.