How to separate proteins of equal size - (May/13/2008 )
Hello. Since SDS-PAGE separates proteins based on size, what modification or methods can i use to separate proteins with roughly the same size? My classmate told me to use native PAGE. Is this correct? Thank you.
native page separates based on charge (with size constraints due to limiting pore size of the gel). if you have two proteins of the same size but different pI then you should be able to resolve them on native or urea or ief page.
so, yes, your classmate is correct.
use better 2D-GE than native PAGE as 2D-GE is more standardized
Is this an exam question? If so, then you can set different situation and give the answer, such as:
1. if only one of them can assemble into homopolymers, then you can crosslink it and run the gel.
2. if only one of them has modification like phosphorylation then you can IP or western using the specific antibody.
3. if only one of them has intramolecular disulfide bond, then you can run non-reducing gel to separate them
for experiment, easy way is IP or 2D gel