4 replies to this topic

[mmolen10]

Hi everybody,

I'm trying to isolate a protein L39 out of ratliver-ribosomes wich we expect to have antimicrobial properties. After a few steps of centrifuging I extracted it the ribosomal proteins with 66% acetic acid. I want to fractionate this ribosomal extract with cat-IEC. Problem is that the proteins after lyofilisation or dialyse don't want to solve in: 50 mM ammoniumacetate pH 7, 50 mM ammoniumformate pH 5, 20% acetic acid, urea 3 M. Any suggestions?

Thanx

[sharath]

Why extactly do you ant to dissolve the protein in 50 mMammoniumacetate pH 7, 50 mM ammoniumformate pH 5, 20% acetic acid, urea 3 M? Why do you feel that lyophilization or dialysis is affecting the solubility?

from what i can undersand about your situation, it could be that the protein is insololuble in buffer you are using. 20% acetic acid is good enough to precipiate a protein. Just 12% acetic acid is used fix proteins after PAGE.

good luck

[mmolen10]

sharath, on Feb 25 2005, 05:06 AM, said:

Why extactly do you ant to dissolve the protein in 50 mMammoniumacetate pH 7, 50 mM ammoniumformate pH 5, 20% acetic acid, urea 3 M? Why do you feel that lyophilization or dialysis is affecting the solubility?

from what i can undersand about your situation, it could be that the protein is insololuble in buffer you are using. 20% acetic acid is good enough to precipiate a protein. Just 12% acetic acid is used fix proteins after PAGE.

good luck

Thnx for you reaction.

50 mM ammonium acetate had the advantage that it's volatile.

I think it wasn't clear in my last post that 50 mM ammoniumacetate pH 7, 50 mM ammoniumformate pH 5, 20% acetic acid, urea 3 M wasn't one solution but several options. So i tried to solve it in 50 mM amm.ac pH 7, in 20% acetoniril and in 3 M urea. None of them solved to proteins. In the 66% acetic acid-extraction buffer they are solved.

Problem is that 66% ac.acid cannot be used for IEC. So i'm looking for alternatives. Important is to know that ribosomal proteins have a relative high pI (about 10-12), so the lower the pH, the better the solvation.

[sharath]

what is the problem with the routine phospahte buffer pH 7.0?

[mmolen10]

sharath, on Feb 26 2005, 07:02 AM, said:

what is the problem with the routine phospahte buffer pH 7.0?

It won't solve the proteins.