4 replies to this topic

[dttom]

I'm confused that there are so many chemicals to add during centrifugation, for example in a lysed cell lysate, some saturated (NH4)2SO4 are added before centrifugation to obtain soluble proteins, but I have no idea why this salt has to be added. Could anyone help?

[Denny]

dttom, on Mar 21 2010, 01:09 AM, said:

I'm confused that there are so many chemicals to add during centrifugation, for example in a lysed cell lysate, some saturated (NH4)2SO4 are added before centrifugation to obtain soluble proteins, but I have no idea why this salt has to be added. Could anyone help?

What are you trying to do?
If you are doing an ammonium sulfate precipitation, you are adding salt, precipitating protein, adding more salt, precipitating protein, etc. You are doing a quick purification of your protein which will precipitate when the salt reaches a certain percentage. The cool thing here is that your protein isn't denatured and when salts are removed, your protein goes back into solution and is still soluble. Then you can further purify it or use it as is depending on what you're doing. Here's a few links with detailed explanations:

http://en.wikipedia....e_precipitation

http://www.encorbio....cols/AM-SO4.htm

Protein Precipitation Using Ammonium Sulfate
Paul Wingfield1

1National Institutes of Health, Bethesda, Maryland
Publication Name:
Current Protocols in Protein Science
Unit Number:
APPENDIX 3F
DOI:
10.1002/0471140864.psa03fs13
Online Posting Date:
May, 2001

Check your PM  

Edited by Denny, 21 March 2010 - 04:43 AM.

[dttom]

thank you for the detail explanation on ammonium sulfate's use on salting out. Indeed, the experiment aims to extract a fraction containing a soluble protein, and the experimental procedure is that, after addition of ammonium sulfate to the cell lysate, the mixture is centrifuged so that the target protein is found in supernatant.
I now think the salt is added to insolubilise (salt out) some soluble proteins so that the supernatant will contain purer target protein.

[Inmost sun]

dttom, on Mar 21 2010, 06:09 AM, said:

I'm confused that there are so many chemicals to add during centrifugation, for example in a lysed cell lysate, some saturated (NH4)2SO4 are added before centrifugation to obtain soluble proteins, but I have no idea why this salt has to be added. Could anyone help?

the salint point is: ammonium sulfate robs proteins the covering water to precipitate WITHOUT denaturation

[sw1232]

dttom,

If you're still trying to salt the protein out of solution just make sure you add the AS slowly with gentle stirring with your protein on ice. I didn't check out the link to the protocol Denny posted, but it should mention those details if it didn't. Adding the AS slowly just prevents locally high concentrations of AS, which could prompt some of your target protein to fall out even as the percent AS is below the threshold for your protein. Sorry if you already knew about this, though.