I'm looking for a good protocol for protein extraction and purification for proteomic analysis. Steps to remove phenols and Rubisco should be included. highly apprciated your help
Dear Saman Seneweera,
In one of my research projects dealing with infection of potato leafs, we spend months testing several sample preparation protocols prior to bottom up proteomics. Like you describe, among others phenols complicated the successful extraction and analysis of proteins. We finally ended up with a working protein extraction protocol followed by in-gel digestion to ensure robustness in the subsequent LC-MS analysis. Please see doi: 10.1016/j.dib.2015.11.069
Hope this helps.
Best regards
Dear Saman,
I think there is no a universal protocol that can be used for purification of all proteins. Each protein have its unique physicochemical properties. So it is very important to develop a purification protocol for each individual proteins. In fact, in some circumstances, one specific protocol can be used for purification of proteins having more or less similar physicochemical properties.
I personally recommend you to develop your own extraction protocol for purification of your target proteins in their soluble and stable forms. You can use a high-throughput screening platforms to identify critical buffer components that can maintain the solubility and stability of your proteins.
You might be able to run your samples over a gel filtration column to remove phenols. Also you might be able to immunoprecipitate Rubisco to remove it. Disclaimer, I have not done much work with plant proteins but I know its hard to extract and purify them.
As Alemu points out, there is no 'universal protocol' for protein extraction/purification. The protocol of choice strongly depends on the kind, origin and amount of your samples. If you want to specify these in more detail you might get a more qualified answer. Alternatively you could do a literature search, of course ;)
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