Dimer or monomer - (Nov/25/2009 )
Good morning, dear all!
How can I easely recognize if my protein is expressed as dimer or as a monomeric protein?
I thik my protein tend ta associate in dimer in presence of palmitic acid acid pruduced by bacteria during expression step.
reagards,thank You very much for the help
fra
Hi fra, u can either run a gel filtration column or native PAGE to determine that....if the observed MW comes out to be double the theoretical one, it could be due to dimerization of the protein....
you can also run sds-page with samples with and without reducing agent.
It would be PAGE, then, not SDS-PAGE ^^
I would go for the native PAGE, +/- palmitic acid. If you're right, your protein will adopt a monomeric form without palmitic acid, and double its molecular weight with palmitic acid. It would look pretty neat !
The most rigorous way of determining oligomerization state of a protein is through equilibrium sedimentation. Maybe you have a core facility where you work which has an ultracentrifuge fitted for sed expers, and you can have a few expers run.
madrius1 on Nov 25 2009, 02:46 PM said:
I would go for the native PAGE, +/- palmitic acid. If you're right, your protein will adopt a monomeric form without palmitic acid, and double its molecular weight with palmitic acid. It would look pretty neat !
no, it is sds-page. you use sds in the sample. the reducing agent is 2-mercaptoethanol or dtt.
never heard of using palmitic acid with native page. sounds intriguing. does the palmitic acid cause a dimer to dissociate? does it break disulfide bonds (as does a reducing agent)?
even i wud do a sds-page (reduced and non reduced).. its the most simplest technique to find out if ur protein is a monomer or in a dimeric form.. native might take a longer time and gfc.. u need hplc.. column etc.. so sds is teh best bet... simplest of all!!!
SDS will most certainly denature protein-protein interactions..
I can hardly see any protein complex resisting SDS negative charging..
madrius1 on Nov 28 2009, 01:28 AM said:
I can hardly see any protein complex resisting SDS negative charging..
yeah, i agree with Madrius.......running a SDS-PAGE will disrupt any interaction and I really don't know how can one see the dimerization in that case.......in fact one of the proteins that i am working wid is trimeric n it does not get reflected on an SDS-PAGE...
n for gfc, why do u need hplc at all????
if the dimer is bonded by disulfide bridges the sds will not dissociate it without reducing agent.
gfc can be run in classical lc columns but hplc is faster and more reproducible (and easier to calculate). it is a good way to look for monomers and dimers with purified proteins.