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Protein Size Exclusion

Protein Size Exclusion - Protein Science

Protein Size Exclusion - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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  #1  
Old 01-26-2010, 04:54 PM
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Default Protein Size Exclusion



Hello everyone!

I need some opinions from you.

I have a protein that forms trimers, dimers, and monomers.
In a SDS-Gel I denature everything and in a Western I just see 1 band.
In a Native Gel, I just can see a monomer...

So, basically, I need a way to separate this forms without denaturate them. (and a cheap metod to do it)
I already try some size exclusin columns. But, in this kind of columns, we are unable to recover the protein that binds to the membrane. We just recover the protein above the cut off

Any ideas?
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Old 01-26-2010, 05:26 PM
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Default Re: Protein Size Exclusion

I assume you can't see a monomer in the native gel.

There is probably hydrophobic interaction causing the protein-protein binding.
Since you can't denature, you need to use buffer components like gentle detergents Tween or Igepal, helper protein like BSA, or casein (provide their own regions for protein-protein binding).

Lastly if the above don't work you can add PEG (PEG is hydrophilic and attaching it to the protein diminishes aggregation and increases solubility).
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Old 01-26-2010, 05:38 PM
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Default Re: Protein Size Exclusion

Also you can try to block all cysteines (which is hydrophobic and has a thiol group that forms S-S bonds) by treating your protein solution with NEM (N-ethylmaleimide). Get rid of excess NEM (if you want to, by putting protein sol'n through PD10 column).

Along the exact same lines, adding reduced glutathione (tri-peptide in size) will also block the cysteines.
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Old 01-26-2010, 07:02 PM
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Default Re: Protein Size Exclusion

Hi!

Yes, I can see a monomer. There is a publication about it.

My problem is, from all celular extract isolate the proteins with high molecular mass like 1000Da. Than, isolate between 1000KDa-500Kda, than 500KDa-300Kda, than 300-100Kda...

That my ideia. After this I can denature all the fractions and run on a gel...
My problem is: how I can I isolate this sizes?
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Old 01-26-2010, 08:52 PM
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Smile Re: Protein Size Exclusion

Quote:
Originally Posted by Arastho View Post
Hi!

Yes, I can see a monomer. There is a publication about it.

My problem is, from all celular extract isolate the proteins with high molecular mass like 1000Da. Than, isolate between 1000KDa-500Kda, than 500KDa-300Kda, than 300-100Kda...

That my ideia. After this I can denature all the fractions and run on a gel...
My problem is: how I can I isolate this sizes?
How about clearing the cell lysate from the insoluble fraction by max centrifugation for 20 min, 4C. Using Supernatant (soluble protein) and running it through a major protein depletion column (like this --> [Only registered users see links. ]).

Then run the eluant throw your size exclusion columns and voila!

The converse method would be to run through an affinity column where you select for the protein-of-interest, wash away all other proteins and then elute the protein-of-interest.
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Old 01-26-2010, 09:21 PM
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Default Re: Protein Size Exclusion

To just "isolate the sizes" try gel filtration, this won't include any sticky membranes.

You can use them with a HPLC or just gravity-flow columns.

For large size separation you probably need superose, but check out your options (like here --> [Only registered users see links. ] and [Only registered users see links. ] )
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  #7  
Old 01-31-2010, 01:04 AM
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Default Re: Protein Size Exclusion

Hello,


Well, the simplest way is to use gel filtration (size exclusion). The big advantages are that you obtain an estimate of the NATIVE relative molecular mass and the protein DOES NOT NEED TO BE PURE. All you need is a reliable assay and a well-chosen gel-filtration resin.

For example, pure lactate dehydrogenase gives a single band on a gel of about 40 kDa (as far as I remember) but gel-filters at a about 140 kDa. As LDH is a globular protein we can tentatively conclude from these data that it is a tetramer. Furthermore, we can obtain an estimate of the molecular weight in relatively crude samples, eg by gel-filtering a brain or testis high-speed supernatant, as there is a very reliable assay for this protein.

This is also very useful for checking that purification has not changed the apparent native relative molecular mass of the target protein


I am surprised that this technique does not work in your case.

Some more basic information would be useful.

1. What is the molecular weight of the band on SDS?

2. What is the evidence for dimer and trimer formation?

3. Under what conditions are you gel-filtering and what gel are you using?

4. What do you attribute losses on gel-filtration to?

One possibility it that it is binding to the column by ion-exchange. Most gel-filtration resins contain a small amount of ion-exchange groups (or so we are told) and for this reason it is usual to include (say) 100mM KCl in the eluant Alternatively, your protein may be binding to the resin hydrophobically, in which case inclusion of 10 percent ethylene glycol may help.

5. What pH are you gel-filtering at and does pH affect the monomer /
dimer/ trimer / tetramer equilibrium?


6 What are the dimensions of the gel-filtration column.

Finally, a caveat. Obtaining any sort of a reliable native molecular mass by gel filtration usually assumes that the protein is globular. Asymetric proteins such as fibrinogen and lysozyme gel-filter anomalously.

Good luck

tgd.


Some useful references (available in PubMed Central) which you probably know about:


Andrews, P. (1964). Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91, 222 - 233.

Andrews, P. (1965). The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595 - 606.
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  #8  
Old 03-08-2010, 05:12 PM
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Default Re: Protein Size Exclusion

Hey.

Very helpfull. I solved it with a gel-filtration column.
I'm able to see isolated monomers, dimers and trimers.

Fantastique!

Thansk a lot
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Old 04-02-2010, 01:08 PM
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Default Protein Size Exclusion

Will you please describe me that which whey protein isolate is best for taking? My weight is 85 KG and I'm dieting to lose my weight. I want to take whey protein but don't know that which brand is good to take...
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