Hello guys, may I know what are the considerations one should have when it comes to the selection of buffers for protein purification? I learnt that optimal pH range and components such as phosphate may interfere with downstream assays. However, I am unsure about the implications of having different types of ion (such as potassium, sodium, magnesium, chloride) in the buffer. I understand that they may be involved in salting-in/ out proteins but apart from this, are there any other considerations that are required for their selection? Thanks.
Adam,
I do mostly Electrospray LCMS work so I'm usually concerned about metal cations being present. So, I'm always looking to use ammonium, as it causes less problems. I'm sure that within every assay, people have their favorites to work with. So, If you have an idea of the types of analyses you want to do downstream, I'm sure you'll get lots of suggestions here.
Not sure what you are trying to do. If you need to increase solubility Arginine is preferable to NaCl. Its often used in SEC column chromatography of antibodies to dissolve aggregates and minimize interactions with the column. However, it will also reduce the affinity of many protein-ligand interactions so it best to make sure its either diluted out for assay or removed. A number of studies have shown that KGlutamate, which is a primary salt in many cells, is a great ion for assays. Particularly nucleic acid/protein binding and DNA enzymes although many other systems like this. A problems with this salt is the opposite of arginine, it seems to promote interactions so at high protein concentrations you may see more aggregation and more sticking to columns. Sometimes a combination of salts can be useful but this is trial and error. In this regard LiCl is often used at low salt. Alternatively, if its a protein stability problem, you can try other osmolytes like glucose, glycerol, trehalose ... or crowding agents such as PEG 1k (there was a recent paper that used this from the Richardson lab at Harvard in one of the Nature Journals, I think Gehard Wagner was also an author).
I would suggest that you read several books on protein purification, front to back. Protein purification is so empirical, yet there are many scientific justifications for many choices. I suggest the Scopes book as a first start, Protein Purification: Principles and Practice. Then, after the concepts from that book are learned, I would get my hands on the METHODS IN ENZYMOLOGY Guide to Protein Purification, 2nd Edition Vol463, and of course download every free application note/handbook from the http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-US/service-and-support/handbooks/ . Be sure you know the Hofmeister series (look it up on Google). Typically, use K salts for intracellular proteins, Na salts for extracellular proteins. There are hundreds of other recommendations. Good luck!
Thank you all for the recommendations and discussions. They have been really helpful.
I agree that the choice of buffer is largely empirical. But you don't have to start from nothing. A good starting point is to look up the literature on the type of protein that you are trying to purify as well as the type of tissue that you are working with. A third point to consider are the purification steps that you are contemplating, and expect that you might have to change buffer as you move from one purification step to the next. Aside from effects of specific ions, the first factor to consider is that you want sufficient buffering capacity to keep the proteins at the pH that you think will keep the targeted protein stable. After you have chosen your buffer components, try out a small batch. If your question is about a buffer for extraction, make an extract in two or three buffers but do it on a small scale and check for activity or the presence of the protein you are looking for. From there you can select one and work up the first purification step. If the protein you are purifying is stable to freezing, always keep small aliquots every step of the way and keep a record of the total volumes from which the aliquots were taken. When you have finished the purification or you have reached a dead end having lost your protein, you can analyze those aliquots and figure out how to change the purification procedure.
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