11 replies to this topic

[FRAROAR]

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

[Vini]

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....

[mdfenko]

you can also run sds-page with samples with and without reducing agent.

[madrius1]

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 !

[StevieRay]

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.

[mdfenko]

madrius1, on Nov 25 2009, 02:46 PM, said:

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 !

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)?

[Prep!]

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!!!

[madrius1]

SDS will most certainly denature protein-protein interactions..

I can hardly see any protein complex resisting SDS negative charging..

[Vini]

madrius1, on Nov 28 2009, 01:28 AM, said:

SDS will most certainly denature protein-protein interactions..

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????

[mdfenko]

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.

[Amit Kumar]

Hi, i tried the following way to determine trimeric nature of my protein and it worked --

1.Go for normal SDS-PAGE but without adding DTT or beta-mercaptoethanol to sample loading buffer.

SDS is only a denaturant agent and breaks only non-disulfide bonds responsible for tertiary andquatnary structure of protein but  DTT or beta-mercaptoethanol breaks disulfide bonds which cuases oligomeric protein to appear monomeric in Reducing (i.e. withDTT or beta-mercaptoethanol) SDS PAGE.
Actually my protein's trimeric structure was due to disulfide  bonds between cysteine residues.

I found all three forms - monomer,dimer and trimer with respective weights

2.I also tried Native PAGE (i.e. without DTT or beta-mercaptoethanol  and without  SDS in gel or any of the buffers) but as Native PAGE separates acco to combination of charge and mass (unlike uniform -ve charge provided by SDS in SDS PAGE and separation acco to size only) so molecular weight of the protein could not be determined even though marker was also run which was not necessary because marker contained DTT and did not correlate with size of protein bands.
u can only see 2 or 3 bands acco to your protein but u cant determine their size in Native PAGE.


I think u should try first method.

[bob1]

Amit Kumar, on 28 March 2012 - 01:17 PM, said:

I think u should try first method.

You should possibly check the post dates on the thread - last replied to in 2009, so i guess the answer has already been found...