Ammonium sulfate (40%) was used to precipitate proteins from calf thymus extraction. After centrifugation, the pellet was dissolved in PBS. The pellet can’t be dissolved completely (still had lots of pellet after centrifuging at 12000g for 10 min).The supernatant was cloudy and couldn’t be filtered through 0.22 um NC membrane. We further centrifuged the supernatant at 12000g for 1 h, but the supernatant was still cloudy and could not get through 0.22 um membrane. To get clear sample through 0.22 um NC membrane is the first step to further purify the proteins. Now it’s totally stucked here. Is there any recommendations? THX.
One possible reason why the pellet could not be fully dissolved is that the volume of buffer added to the pellet was insufficient to lower the residual ammonium sulfate concentration below the level at which the protein would redissolve. Adding more buffer may be all that is needed.
Dear Di Fan
You can have a problem with nucleic acids. If your protein does not bind to nucleic acids, you can remove them by "salting out" with streptomycin sulfate. I used a 2% final concentration. After adding streptomycin sulfate, you centrifuge a solution and take a supernatant for further steps (i.e., ammonium sulfate precipitation).
It is also important to centrifuge a lysate before all further steps to remove not dissolving molecules (lipids, etc.).
Regards
Re-suspension: Try re-suspending the pellet in a smaller volume of PBS or a different buffer that is compatible with your downstream purification method. Gentle stirring or agitation might help in breaking up the pellet. Also, ensure that the PBS is at the correct pH for your protein of interest.
Buffer Adjustment: If PBS is not effective, consider adjusting the pH or ionic strength of your buffer to enhance solubility. Sometimes, small changes in buffer conditions can make a significant difference in dissolving the pellet.
Protease Inhibitors: Add protease inhibitors to your buffer to prevent protein degradation during the dissolution process. Protease contamination can cause cloudiness in your solution.
Detergents: In cases of hydrophobic proteins, adding a mild detergent like Triton X-100 or NP-40 to your buffer can help solubilize the proteins. Be cautious with the detergent concentration, as excessive detergent can interfere with downstream applications.
Extended Centrifugation: If you're unable to clarify the solution through a 0.22 um membrane, try a longer and higher-speed centrifugation step. It's possible that some precipitates or contaminants are still present.
Filtration: If centrifugation doesn't clarify the solution, try using a lower pore size filter, such as a 0.45 um membrane filter, before attempting the 0.22 um filter. This may help remove larger particulate matter.
Alternate Precipitation: Consider using an alternative protein precipitation method like acetone or trichloroacetic acid (TCA) precipitation, which can sometimes result in a cleaner protein pellet.
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