Hello everyone,
I just want to have a quick question (and hopefully a quick and brief response), what are the advantages and disadvantages of different protein purification methods, say ionic precipitation (i.e. ammonium sulfate precipitation, LiCl precipitation, TCA precipitation) for determining maximum protein quantity and quality?
Can someone make a brief comparison of the different techniques used for protein purification, as well as their advantages and disadvantages over the other? Please also include limitations of the process and technology. If you have supporting papers, please include them here.
Thanks in advance.
This really very much depends on the material from which you are extracting the proteins, and how "dirty" that material is.
The two links below compare some extraction techniques, one in plants, one in food.
In short, and in my experience, the precipitation methods are more or less equally useful for "easy" samples. I have extracted many samples originating from poplar leaves, and it works just fine to grind the leaves to a powder and precipitate the proteins in the buffer (followed of course by washing etc.)
Oak leaves on the other hand are rich in secondary metabolites (mostly phenols) and require a pre-purification with phenol. This can also be true for roots, tubers, or other tissues (not necessarily plant) rich in secondary metabolites, fibres, phenols, long chain hydrocarbons, basically anything that would co-precipitate with the proteins.
I have also extracted proteins from flatworms, by first dissolving the worms in hot SDS, followed by precipitation. This worked marvellously. You just have to make sure that you wash the samples very well, since the SDS can interfere in techniques like isoelectric focusing.
Which brings me to my final point. It depends on what you want to do with the proteins, since precipitation techniques generally denature the proteins.
Here a limited selection of recommended literature.
Hope this could help.
http://www.davidpublishing.com/davidpublishing/upfile/10/31/2012/2012103165959257.pdf
http://link.springer.com/chapter/10.1007%2F978-1-4614-5626-1_2
Protein purification methods are many and you have specifically mentioned the salting out/precipitation of soluble. These are non-specific methods which exploit the solubility difference of proteins present in a mixture. Salting out by ammonium sulfate precipitation results in a precipitate with a high salt concentration which needs to be dialysed out. However, this is easy and cheap methods. Precipitation by TCA is quicker but has the disadvantage of having limited use. TCA is also a hazardous chemical.
Hope this helps,
The protein purification methods need to be applied depending on your protein characteristics and particularities. It will depend also in your downstream analysis.
So, i recommend you to search in GE health science website (or other source) in the "handbook" section, cause they have good free-books about the most common techniques for protein purification and analysis.
Quantity of protein: usually you can do a Bradford assay or use a Nanodrop in the wavelength of 280nm (but you will need your protein molecular extinction coefficient).
Quality of protein: usually you may have an idea of quality of you purification in the purification processes (e.g. Size Exclusion Chromatography) but is recommended to do an enzymatic (if your protein is a enzyme) assay to verify if your protein is still active.
As you may see, there is no "short" answer. :(
However, you can find very good (and fast) explanations around.
Some youtube videos, as examples: https://www.youtube.com/watch?v=7h0XrF1BleM
affinity chromatgraphy (IMAC) - https://www.youtube.com/watch?v=FUAQKjKT99Y
However, do not rely only in these short explanations. Try always to "RTFM" (read the fucking manual). There are very specific and detailed pros and cons, when and how to use the techniques.
Sorry for the not so short answer
Good lucky
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