Hello,
I am trying to figure out what is the use of the Amicons. I was idealistically hoping that it will help me separate proteins based on their molecular weight. As it can be seen in the image below, this is not the case. I used a 50k cut-off Amicon/Microcon and the flow-through contained no significant amount of protein, while in the concentrate all the proteins are to be found in the same proportion as in the original protein mixture. See image below (the first lane after the LMW is the mixture before applying it onto the Amicon; following flow-through at 30k and concentrate at 30k; flow-through at 50k and concentrate at 50k).
If nothing happens differently at 30 and 50k cut-off, how is the cut-off to be chosen? Can the amicons be used at anything else except for buffer exchange and concentration?
Thank you in advance!
Welcome to reality. MWCO's are far from what you are thinking. They refer to perfectly spherical proteins and still some proteins will pass. Anyway I hope the gel was run under native conditions. Amicon filters will always let protein pass by to a different extent, the further the molecular weight of the protein from the nominal MWCO the greater the rejection coefficient. My decision to start testing membranes is to consider that the membrane will retain protein two times larger than the molecular weight cutoff.
A far better method to separate proteins by molecular weight is gel filtration chromatography.
MWCO filters are used to concentrate proteins. The pore size is chosen to avoid loss of the protein of interest through the filter.
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