I have precipitated crude proteins from extracellular media of a bacteria with up to 80% ammonium sulphate precipitation. I'm performing dialysis to desalt the protein sample in 1X PBS pH 7.5. After 2-3 exchange of 4 hour each, the protein sample starts precipitating. What could be the reason.
Not all the proteins dissolve completely in buffers and some times after dialysis, proteins may precpitate at the bottom.
It happens in case of highly glycosylated proteins as well, so if your protein is glycosylated , it's solubility in the buffer being used is being affected .In which case lyophyllization wouldn't help. Slowly change the ionic strength of buffer and it's pH ,one by one and optimize the buffer to be used for dialysis.
Currently I'm not sure it is only protein which is precipitating because I have not run SDS-PAGE for these precipitate, but I'm not figuring what could else be possible if these ppts are not proteins, because after ammonium sulphate salting out there should be only proteins.
Dear Sandeep:
First, check your sample by SDS-PAGE. If you have proteins in your precipitate then it looks quite clear to me that you have halophilic-like proteins. They didn't precipitate during ammonium sulphate precipitation but as soon as you desalt it by dyalisis, proteins showing halophilic pattern precipitate.
Best regards,
Rosa María
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH