i have precipitated enzyme by acetone.. when i use coomassie staining in native gel electrophoresis, it couldnot migrate to stacking and then resolving gel. is there is a problem in acetone precipitation ? as when enzyme resuspension in buffer after precipitation, there is turbidity..
Acetone has likely denatured your proteins and native gel electrophoresis requires soluble proteins. Try a denaturing PAGE or maybe a urea gel. MWCO spin filters can sometimes concentrate proteins enough for electrophoresis without harsh precipitation, or other types of precipitation may keep proteins soluble. Good luck!
There is a technique called Blue Native PAGE. Basically, it is like Native PAGE except electrophoresis happens in the presence of Coomassie Blue-G250. You electrophorese you gel in this blue buffer.
Basically Coomassie Blue-G250 gives proteins a negative without denaturing your protein. So your protein is simply folded as it runs through the gel, except it has a negative charge while still being folded.
https://blog.benchsci.com/principles-of-blue-native-page
@Edward, what is other precipitation methods that make protein soluble
It is unlikely that the problem with your enzyme migration is due to acetone precipitation. Acetone precipitation is a common method for protein purification and is generally considered to be a reliable method. The turbidity that you observe upon resuspension of the enzyme in buffer is likely due to residual acetone, which can interfere with protein quantification and downstream applications.
The problem with enzyme migration in native gel electrophoresis could be due to a number of factors. One possibility is that the enzyme is too large to migrate through the stacking gel. Stacking gels are typically designed to focus smaller molecules and can have limited ability to resolve larger molecules. Additionally, the pore size of the gel may be too small to allow the enzyme to migrate through.
Another possibility is that the enzyme is not sufficiently denatured or lacks sufficient negative charge to migrate through the gel. In native gel electrophoresis, proteins are not denatured and retain their native structure, which can limit their migration through the gel. Furthermore, if the enzyme has a net positive charge, it may not migrate through the negatively charged gel.
To address these issues, you may want to try denaturing the enzyme by heating it in a sample buffer with a reducing agent prior to loading it onto the gel. This can help to overcome issues with size and charge, allowing the protein to migrate more effectively. Additionally, you can try using a different type of gel or modifying the buffer conditions to optimize enzyme migration.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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