I work on downstream processing. my question is whenever i do ammonium sulphate fraction i get floating pellet but, not getting settled pellet. what wrong in my protocol. I add 50 ml 100 % ammonium sulphate solution to 50 ml of fermentation broth? incubate overnight @ 4 degree celcius. please any one help my doubt
I think you are adding the ammonium sulfate fast, if so, it will create a uneven distribution of the salt stabilization and leads to denature of proteins and the denatured protein will float on the surface (your floating pellet). The reason to add ammonium sulfate slowly with constant mixing is, this salt will acidify the solution which facilitate the precipitation of proteins. If you add the salt really fast thet you end up creating uneven pH distribution leading to denaturing the proteins. You can also try with different ammonium sulfate saturation (30%, 50%..70%) and purify the proteins after each step, which will give you an idea to decide which saturation concentration to go with.
If this downstream process is for fermented medium use cell free culture broth.
Read the more information in the following links.
http://web.mnstate.edu/provost/AmmoniumSulfateProtocol.pdf
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/SAFC/Bulletin/t040.pdf
http://hymanlab.mpi-cbg.de/hyman_lab/wp-content/uploads/2012/07/Ammonium_Sulfate_Precipitation.pdf
i too agree with both the answers....first u have to the optimum saturation level of salt required for the precipitation of ur targeted protein. To do so u need to add salt slowly (powder or liquid) to the broth and allow for mixing and incubation. after that u need to do centrifuge minimum at 10000 g. i hope by folliwing these things u will get good results....
all the best
Follow the link below to calculate the exact value of amm. sulphate for saturation calculation. I was benefited a lot.
There should be no precipitate or debris floating in the solution before addition of ammonium sulfate. If required, centrifuge at above 10,000g at 4 degree cel. for 10 minutes.
http://www.encorbio.com/protocols/AM-SO4.htm
Dear All,
I have the same problem of pellet floating even i let teh AS precipiation more than one hour ,where is the problem ?
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