What is differences between ion exchange and size exclusion chromatography based on yield and purify. Which one has higher purity/yield and reliable?
Dear Tuğçe
The answer to your question is not easy becuase it depend a lot from the properties of the sample that you would like to purify.
Generally ion Exchange is more used as first step of purification in absence of tags which allow affinity purtification, as IMAC, MBP, GST, proteinG/A affinity purification, while SEC is more used as a second step to refine the purification, remove possible aggregates and buffer Exchange the sample in a single step.
Good luck
Manuele
in size exclusion chromatography compound separated by its size and in some case by its molecular wight while in case of ion exchange chromatography ions separated according to infinity with ion exchanges.
Hi there,
IEC is much more resolutive because conditions for elution can be optimized (nature of the matrix, buffer pH, nature of the salt, length and slope of the gradient) whereas it is not the case for SEC (isocratic elution).
To answer your question it is little bit tricky because we don’t know what is the composition of your sample (e.g sample contains expressed protein with affinity tag or sample contains mixture of proteins isolated from animal or plant source, etc) If the sample contains expressed protein with affinity tag then you should check the purity of your protein after expression by using SDS-PAGE, if the SDS-gel shows big expressed protein band and few low or high molecular weight impurities then you should run size exclusion column but if the SDS-gel shows multiple bands then you need to try other methods of separation like IEC, HIC and you can optimize different parameters like pH of buffer, nature of salt, type of resin during these separation methods. Finally if your yield of protein is low then don't go for SEC because you will lose 30-40% of your sample during the process.
The best method for purification is depending on the properties of the target protein and the properties of the other proteins in the sample and their quantity .
Usually you can use ion exchange for proteins with differences in their IEP . Size exclusion chromatography will used to separate proteins with diffuseness in their M.W.The quantity of sample you can separate in IEC is higher than size exclusion chromatography .But usually you most use more than 1 type of chromatography to purify protein.
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