Hi,

If you want only to see the different conformations, the the non-reductive gel can help you. I have done it, and it got right.

How your SEC purification is going on? In some cases, it is possible to separate the oligomeric states of a protein by selecting the correct column and time for SEC purification.

Tell me if you need anything more.

I'll send a pic of non-reductive gel I've done.

Observe in the pic that I was working all the time with a protein that had 27 kDa. When I performed a run with a non-reductive gel I observed that the protein had dimeric conformation.

It is important to highlight that the hexameric conformation might be hard to see in the gel, even in these conditions, because of the size of the protein. Try different concentrations of poliacrilamide. Hope it could help you.

Sincerely,

Pamela

Dear Sitara

theoretically sds page is denaturing and oligomeric form are lost and you see dimers ir oligomers in absence of reducing agent only if the dimerization/oligomerization is mediated by s-s bonds formation which is resistant to sds.

Practically it depends a lot from the stability of your hexamer- dimer. some very stable proteins are able to mantain Its quaternary structure during sds page (if you not boil the sample and not add dtt) but this are rare exceptions (it is happened to me for human Cu,Zn Sod1 protein)

in the other cases you need to run blue native elecrophoresis.

https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/nativepage-bis-tris-gels.html

or replace sds page with size exclusion chromatography.

you can find some examples of reducing and non reducing sds page on my blog: https://proteocool.blogspot.com/?m=1

ProteoCool n 15: Overwiew on SDS page

ciao

Manuele

Thank you very much Manuele Martinelli and Pamela Pena for your kind suggestions. I have tried reductive SDS PAGE, Non- reductive SDS PAGE and also native PAGE. I got a single monomeric band i.e. 45 kDa in case of reductive SDS PAGE, which makes sense as DTT would have reduced the cysteine residues. With Non- reductive SDS PAGE, target protein crossed the stacking gel but unable to move in resolving gel might be due to its hexameric conformation which makes it size around 200 kDa, but does this make sense that all the protein subjected to refolding is successfully refolded into hexameric conformation as I did not get any other band i.e. of monomeric form (45 kDa) or dimeric (80 kDa) form which are reported otherwise. Lastly in native PAGE I did not get any satisifactory results and I was just curious why people perform this?

Moreover, now I am trying to further purify oligomeric states of target protein through SEC using superdex 200 increase 10/30. Have you any idea which protein standards should I use, or any other guidance related to SEC would be appreciated.

Thanks again!

Dear Sitara

i'm tappe that you find usefull our suggestions.

if your protein cone from revolving i think is it better confirm of the aggregate that you are in absence of reducing is really your hexamer or is it an higher mw aggregate formed during refolding due to aspecific interaction of cys because refolding if protein with many cys is something challenging.

Sec could be a solution because supedex 200 is able to separate up to 600KDa proteins and your hexamer is about 270kda.

Ge sell some calibration kits i think that hmw can be ok for you.

One other simple possibility is run a tris-acetate 3-8% non reducing sds page which is able to discriminate proteins in this mw range.

regarding advice for sec. equilibrate the coloumn with the protein buffer (presence of Salt at least 150mM is preferable) load small volume (100ul) of sample (minumin concentration depends from protein estimation coefficient if you follow it at 280nm) however load at least 100uM of the monomer of you can.

to load so small volume you need to use an Hamilton syringe and the meyql needle port instead the standard sample loading port.

https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/nupage-tris-acetate-gels.html?s_kwcid=AL!3652!3!222062075935!b!!g!!%2Btris-acetate%20%2Bgels&ef_id=CjwKCAjwmq3kBRB_EiwAJkNDp7CL8-ZRpf7Zz5NClINN3IpCiquSS__JNwarLT0f1kJAuwhbUkq5ihoCpEEQAvD_BwE:G:s&s_kwcid=AL!3652!3!222062075935!b!!g!!%2Btris-acetate%20%2Bgels&gclid=CjwKCAjwmq3kBRB_EiwAJkNDp7CL8-ZRpf7Zz5NClINN3IpCiquSS__JNwarLT0f1kJAuwhbUkq5ihoCpEEQAvD_BwE

good luck

Manuele

I agree with Manuele Martinelli's excellent advice. A protein stuck at the stacking/resolving gel boundary suggests the presence of aggregates larger than just a 200 kDa hexamer, which would certainly enter the gel (I have seen people working with > 400 kDa proteins and those don't get stuck there). Using electrophoresis to judge polymerization status has the inconvenience that sample preparation (heating, etc) may actually promote aggregation; people continue to use it only because it's convenient and fast.

Thank you very much Manuele Martinelli for your useful suggestions. I will definitely follow them. Thanks again!

To determine oligomerisation by electrophoresis you need to avoid both reducing agents and detergents. Some proteins run well under these conditions, but some don't. You can use gel systems with different pH (for example doi:10.1002/elps.1150040303 or doi:10.1111/j.1749-6632.1973.tb47551.x). Alternatively, you could try blue native gels (BN-PAGE, doi:10.1006/abio.1994.1112).

But gel filtration, analytical ultracentrifugation, fluorescence polarisation or small angle scattering of laser light, X-rays or neutrons provide alternative routes.