Using PEG/salt aqueous two phase extraction system, after the partitioning of protein to either the polymer or salt phase, which are the suitable methods for concentrating of these proteins?
Any effective and simple methods other than the dialysis approach?
Ultrafiltration is the fastest and simplest. Amicon filters will work for the salt phase and for the other phase they will work if the PEG size is different from the protein (looking at the hydrodynamic radii)
Thanks, Dr. Omar for your reply
Can the TCA or ethanol precipitation method work in my case?
Yes you can induce protein precipitation. I am not sure if it will work for the peg-rich phase and the protein must resist the treatment.
Great thanks, Dr. Omar for your help. I will make a trial with it.
I agree with Dr Omar. Ultrafiltrafion should work. I used both amicon and vivaspin concenterators using them to concentrate and replace the buffers with removing high concentration of salt. Please note that the use of cut-off point close to the size of protein can lead to its loss. For example, if I want to concentrate IgG from blood serum, I use the 50 (or 30) kDa filter instead of a 100kDa.
My protein partitioned to Poyethylene glycol phase in aqueous two phase extraction. now i want to separate protein from PEG . Iam thinking of going to ultrafiltration as litrature suggests it can possible. My protein molecular weight is 50kDa and polymer weight is 6000 . in my lab i have to use disk type ultrafiltration membrane. how much molecular weight cutt off of ultrafiltration membrane i have to select.
You need to know the real size of the PEG molecule, I am pretty sure it will be close to the size of the protein; thus ultrafiltration might not work.
PEG Molecular weight is 6000D and protein is 50000D.....is it possible to choose a membrane which will retain protein and allow PEG to pass through........
I am using peg 4000 for partitioning of my protein which is around 55 KDa. I am also having the same problem as Rashmi has. Please suggest some remedy.
As I said molecular weight comparison is irrelevant. Look for the real sizes to determine if there is significant difference such that a membrane can be used for separation.
If there is no significant difference (as I suspect) you could use ion exchange chronatography if conditions are appropriate.
The radius of your protein will be approximately 2.4 nm (Erickson, Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy) while the PEG molecule has an approximate radius of 2.5 nm (Fee and Van Alstine, PEG-proteins: reaction engineering and separation issues)
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