While doing extraction of proteins by using ammonium sulphate as salt system, and if it is left to be in salt does it affect the protein nature such as denature, activity lose, changes in functional groups?
I hope you are concerned about the prolonged effect of Ammonium Sulfate preparation on the proteins. Ammonium sulfate is a non inactivating protein precipitant and stabilizer which works on the principle of salting in and salting out (solubility). Several commercial protein compounds like enzymes are ammonium sulphate suspension. Ammonium and sulphate both belong to the Hofmeister series being the most stabilizing ions. I would not bother much if I have stopped the extraction for a while and want to start it again from the same. There are lots of available literature if you look for it. Hope it was useful.
I hope you are concerned about the prolonged effect of Ammonium Sulfate preparation on the proteins. Ammonium sulfate is a non inactivating protein precipitant and stabilizer which works on the principle of salting in and salting out (solubility). Several commercial protein compounds like enzymes are ammonium sulphate suspension. Ammonium and sulphate both belong to the Hofmeister series being the most stabilizing ions. I would not bother much if I have stopped the extraction for a while and want to start it again from the same. There are lots of available literature if you look for it. Hope it was useful.
Ammonium sulphate is commonly used to precipitate and store proteins for long standing, usually is completely innocuous and preserves the native state of proteins. Many enzymes are sold as ammonium sulphate suspensions.
can i use this Ammonium sulphate in the protein lysis buffer (SDS or TritonX ) for SDS-PAGE gel? and what is the preparable concentration ?
Hi Rushendhiran, may I know why do you need to use AmSo4 in lysis buffer? If all you need is stability of your protein then I suggest you to add upto 10% glycerol in your lysis buffer.
It does not denature the proteins. If high amounts of salt are present during the assay, it could affect its activity, but you can remove the salt by dialysis/spin column. When you make the ammonium sulfate solutions, if you pH it, you can maintain the pH where your protein is stable. If the pH changes, because of added ammonium sulfate, this could denature your protein. You don't loose functional groups.
High concentrations of ammonium sulfate may affect the way the protein runs in the SDS gel.
Thanks a lot for your valuable commands .i had this doubt ,it can affect the protein functional (phosphorylation site) property.
Precipitation by various salts or nonionic polymers is the preferred method to utilize whenever possible. These precipitations typically yield stable non-denatured
products. also there will be no alteration in protein functional (phosphorylation site) unless denaturation of the protein is not a concern during precipitation.
While doing ammonium sulfate precipitation, you have to take care of the percentage of ammonium sulfate used and off course temperature 4 degree celusius is maintained to retain the activity.
Concentration of ammonium sulfate will no way denature your protein making sure that you optimize and keep the reaction conditions perfect.
Although ammonium sulfate precipitation is currently a relaible procedure in terms of enzyme activity and protein structure recovery one cannot exclude that very high AmmSulf conc could lead to full dehydration of some proteins having water in their structure (eg collagen) thus resulting in irreversible denaturation. As well irrevesible aggregation might possibly occurr at precipitation, irrespective to AmmSulf .
could i store my ammonium sulfate precipitation solution for many days? prior to centrifugation in creating a protein pellet?
Can I store my ammonium sulfate precipitation solution for 48 - 72hours?
If your protein is buffered sufficiently well the addition of AMS will not denature proteins. However AMS solutions are slightly acidic and at high concentration it may denature and irreversibly precipitate some proteins if added to weakly buffered proteins sensitive to pH changes.
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