Extract from a plant has too many proteins. If I perform ion exchange as an primary purification process and then apply these fractions to be seperated based on their molecular size, I will have many fractions in hand. Can I proceed with above said technique?
In addition to Matteo's suggestion, I would do sepharose G-25 column buffer exchange before applying the extract for Ion-exchange. Another way of going about it is using Gradient ammonium sulfate precipitation to get the graction with your protien and the do ion-exchange or size exclusion.
It depends on what your aim is. If you are looking after purifying a specific protein from your crude extract then you will need to investigate the characteristics of that given protein (eg: isoelectric point, molecular weight) so that you will be able to select a chromatographic technique/conditions suitable for the purification.
If your aim is to characterise the protein profile of an extract in general, the two step chromatography that you have in mind could work. you would obtain a pre-fractionation by IEC followed by molecular size separation. To make sure that you are having an appropriate separation you can run electrophoresis on your fractions, as well as RP-HPLC in order to visualise differences in protein profile.
you could also do the other way around, so run SEC first as a way to obtain a broad separation of proteins based on MW, and then another type of chromatography as cation or anion exchenge.
Cheers
Matteo
In addition to Matteo's suggestion, I would do sepharose G-25 column buffer exchange before applying the extract for Ion-exchange. Another way of going about it is using Gradient ammonium sulfate precipitation to get the graction with your protien and the do ion-exchange or size exclusion.
Hi Prasanth,
As Matteo said, If you are looking for a specific protein, you will need to investigate its characteristics and then to find where it is eluted. Maybe it will be necessary other steps.
However, if you want study the protein profile (proteomic study), you will have to do a reversed phase chromatography (C18 column) or a 2-D electrophoresis with the extract to identify the families of proteins
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