Question

You run an ion exchange column, eluting your protein with a high NaCl buffer. Would you be able to run a size exclusion column immediately after the IEX column or would you have to dialyze your sample to change the buffer first?

Answer 1

Dear friend the answer is

yes you would be able to run a size exclusion column immediately after the IEX column. because A small amount of salt will help prevent nonspecific interactions between the protein and stationary phase. This will allow all protein molecules to run consistently over the length of the column in SEC (Size exclusion chromatography).

It is often best to run IEX-HIC-SEC. When you start with IEX (ion exchange chromatography) and then intend to run HIC(Hydrophobic interaction chromatography), it is best to measure the conductivity of the pooled fraction after IEX. Then add salt to that fraction until it has the same conductivity as the start buffer for HIC. With a complex sample like an E. coli lysate, there is a risk of precipitating protein when the salt concentration is increased to the level required for HIC. It is not uncommon that the purification objectives are reached after affinity chromatography and SEC(Size exclusion chromatography), and then there is no need for an intermediate step.

SEC (Size exclusion chromatography) separates proteins by their hydrodynamic radius, a property determined by both the size and shape of the molecule. Unlike the other chromatographic procedures described previously, proteins do not bind to a stationary phase in SEC. Instead, proteins are separated by the speed at which they navigate through an inert stationary phase. A single solvent is used throughout the entire SEC procedure, and commercially available SEC stationary phases are compatible with most commonly used buffer components.

Thank you.