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Dimer or monomer


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11 replies to this topic

#1 FRAROAR

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Posted 25 November 2009 - 07:25 AM

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

#2 Vini

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Posted 25 November 2009 - 07:56 AM

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....

#3 mdfenko

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Posted 25 November 2009 - 08:38 AM

you can also run sds-page with samples with and without reducing agent.
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#4 madrius1

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Posted 25 November 2009 - 11:46 AM

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 !

#5 StevieRay

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Posted 26 November 2009 - 04:56 AM

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.

#6 mdfenko

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Posted 26 November 2009 - 09:54 PM

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)?
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#7 Prep!

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Posted 26 November 2009 - 10:04 PM

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!!!
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#8 madrius1

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Posted 27 November 2009 - 10:58 AM

SDS will most certainly denature protein-protein interactions..

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

#9 Vini

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Posted 28 November 2009 - 05:29 AM

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... :lol:
n for gfc, why do u need hplc at all????

#10 mdfenko

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Posted 28 November 2009 - 08:08 PM

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.
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#11 Amit Kumar

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Posted 28 March 2012 - 01:17 PM

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

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 weightsPosted Image

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.

#12 bob1

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Posted 28 March 2012 - 07:46 PM

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...




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