I have to purify a protein which turned out to be in "Insoluble" fractions when an expression check is performed.
I would be using a denaturing approach with 8M Urea followed by dialysis and the refolding by 0.5M arginine.
If I use my protein pellet within the dialysis bag, is there a possibility that the 0.5M arginine containing buffer can be re-used when I purify this protein again?
I would be greatly helped with a suitable suggestion. I have attached the protocol as well.
Thank you.
I think is do not indicated the reuse of the arginine solution. This buffer would be progressively enriched by denaturant (urea).
Since you removing urea in first dialysis step there is no problem in reuse of arginine solution.
Although there will be slightly lower concentration of arginine in refolding buffer for the subsequent cycles this will not have any significant impact on quality of refolded protein.
I would worry about the arginine as a good C- and N-source for microorganisms.
I don´t understand your refolding protocol. Usually you dialyze the solubilized protein directly against refolding buffer to allow refolding from the soluble protein. Doesn´t your protein precipitate again upon urea removal in absence of arginine or does it already refold/ stay solubilized in your buffer X?
I would refrain against re-using the 0.5 M Arginine buffer for a variety of reasons.
1. The urea present in the protein solution moves to the Buffer
2. Storing the used buffer is prone to contamination (fungal)
I have used arginine refolding extensively and i can say that it works pretty well. Refer to my article in case you have doubts.
Bundle of thanks for the answer.
We need to isolate proteins for NMR spectroscopy, those would be labelled.
I need to re-use the solution because for ~1gm insoluble protein, it would take liters of 0.5M arginine and still unconfirmed recovery percentage.
Even though you might require litres...i would still refrain from re-using the solutions. Im not sure about the cost of a kilogram of L-Arginine in Pakistan. However we in India dont find it too expensive.
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