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Protein precipitation

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Protein precipitation

Postby Miss_Me » Sun Mar 04, 2007 4:52 pm

Hi. Can someone help me out here please!

Which is the best method to recover a precipitated protein back into its normally folded state?
The following solutions were used:


-a- 95% Ethanol
-b- 25% Ammonium sulphate
-c- Solid ammonium sulphate
-d- 5% Trichloracetic acid
-e-10%Perchloracetic acid
-f- 2.5% Perchloracetic acid
-g- 10%w/v sodium tungstate in 0.2M H2SO4
-h- Uranyl acetate
-i- 0.15M Barium hydroxide and 5% zinc sulphate


At first I thought that it could be -g- but am having second thoughts. Could it possibly be -h-? I'm thinking this because uranyl acetate would form a insoluble complex with heavy metals, thus meaning that the structure of the protein would not have changed causing it to denature. The other solutions would cause a structural change in the protein causing it to denature.
Am I right in saying this?


NB. the protein being spoken about is albumin.


Thanks!!
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Postby Dr.Stein » Mon Mar 05, 2007 7:42 am

Statement 1:

"...The precipitation of albumin is completely reversible with respect to pH. An albumin precipitate formed can be completely dissolved by making the solution somewhat alkaline (pH 6 to 7) to the pH at which precipitation occurred..."

For the first step we must eliminate non alkaline/acid things. Now we have this:

-a- 95% Ethanol
-b- 25% Ammonium sulphate
-c- Solid ammonium sulphate
-g- 10%w/v sodium tungstate in 0.2M H2SO4
-i- 0.15M Barium hydroxide and 5% zinc sulphate

Statement 2:

"...Most of the experiments were made with sodium salts. among which sulphate, phosphate, acetate, citrate, tartrate, chromate, chloride, nitrate, and chlorate formed a series arranged in descending power of precipitation..."

Now we have this:

-a- 95% Ethanol

;)


Note:

Some of acids will precipitate albumin, the recovery depends on what made it precipitate:
- Trichloracetic acid ---> the precipitation will be recovered in organic solvents i.e. ethanol, acetone
- Perchloric acid, Benzenesulfonic acid ---> the precipitation will not be recovered in organic solvents
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Postby Miss_Me » Mon Mar 05, 2007 11:03 am

Thanks.


But after i put up the question, i went back to working it out. And so i came up with this:

"Barium hydroxide would be a possible method to recover precipitated proteins because there would be a chemical reaction with the proteins as aDsorption would occur on to the precipitate"

aDsorption means that the there is attachment to the surface unlike aBsorption involving it to be taken in. Thus, this could be a method..right?


Thanks again!!
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Postby Dr.Stein » Tue Mar 06, 2007 8:00 am

I think barium hydroxide, barium acetate, barium citrate, and some other acidic barium are used to precipitate albumin instead of to recover it? Barium will attach and cover albumin molecules surface (yes, it is adsorption), so that it cannot dissolve/being precipitated? :?
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Postby Miss_Me » Tue Mar 06, 2007 10:09 am

yes, barium hydroxide would be used to precipitate the protein.

Would i be correct in saying that if i used each of these solutions separately (in different test tubes) in order to precipitate the protein then if i wanted recover the protein back then it would be easierst to fully recover the protein back from the barium hydroxide solution?



Thankss for your help :D
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