1 reply to this topic

[Hadi]

I would like to precipitate entire protein content of supernatant for further examinations. Activity of proteins after re-suspending is important. I need some advise in the following topics with attention to the above mentioned concerns. Any help would be fully appreciated.

• Should I use the highest concentration of ammonium sulfate gradually in one step to precipitate all proteins or it may has adverse effect on low soluble bigger proteins activity?
  
• Regarding to save protein activity, which one is preferred? adding solid A. S. or saturated solution.
  
• Whether solid ammonium sulfate denature proteins or not?
  
• Is this method able to precipitate small peptides like cyclic dipeptides?
  
• Is it able to precipitate lipo-peptides and peptidoglycans too?

Regards - Hadi

Edited by Hadi, 27 May 2011 - 12:18 AM.

[mdfenko]

Hadi, on 27 May 2011 - 12:14 AM, said:

I would like to precipitate entire protein content of supernatant for further examinations. Activity of proteins after re-suspending is important. I need some advise in the following topics with attention to the above mentioned concerns. Any help would be fully appreciated.

• Should I use the highest concentration of ammonium sulfate gradually in one step to precipitate all proteins or it may has adverse effect on low soluble bigger proteins activity?

that's how i would do it. add a.s. slowly, with cooling and gentle stirring and allow to precipitate, in cold room or on ice for some time to enhance precipitation.

Quote

Regarding to save protein activity, which one is preferred? adding solid A. S. or saturated solution.

i've used both. using solid will avoid very large increases in volume. using a.s. solution to get to high concentrations will cause large increases in volume.

Quote

Whether solid ammonium sulfate denature proteins or not?

if added slowly and gradually then it won't be any worse than adding solution

Quote

Is this method able to precipitate small peptides like cyclic dipeptides?

in general, no

Quote

Is it able to precipitate lipo-peptides and peptidoglycans too?

it should. we us it for glycoproteins without any problems.

you may want to have a look at this protocol from cold springs harbor:

Attached Files

•  precipitataion of proteins by as.doc   215K   680 downloads