Hi, I'm attempting to determine the conjugation efficiency of a reaction between nucleic acid aptamers and PEG-maleimide. My idea was to separate free aptamer from the conjugated PEG using a centrifuge filter (Pierce Protein concentrator) then measure with Nanodrop. The aptamer MW is 3,925.5 Da and the PEG is 20 kDa. I've been using a 10 kDa MWCO filter but I'm barely getting any volume in the filtrate. When I measure the concentration of the retentate my values are much larger than expected presumably due to the conjugated aptamer-PEG skewing results as UV spec is for free nucleic acids. Does anyone have any experience using a Pierce protein concentrator or know a better method for separation that like a syringe filter maybe ?
Thanks in advance
MWCO is generally reported for the size of protein molecules. Moreover PEG size does not translate as protein size based on molecular weight. Furthermore PEG molecules are used as non-fouling molecules, thus they will affect the filtration process. Look into ion exchange. I suppose you have PEG in excess.
Omar Gonzalez-Ortega Thanks for the insight. I suppose this is due to the coil behavior of polypeptides. Yes I have PEG in excess
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