I am isolating exosomes from human plasma using the IZON SEC column. I want to detect exsomal markers Flotilin-1, CD9, HSC70 and TSG101 in my samples. I am using SDS -Page and PVDF membrane, all pretty standard. As a positive controll I am using human monocytes. Before loading on the gel I add 4x laemmli buffer and cook my sample at 95° for 10 minutes.
So far I can only detect Flotilin-1 reliably both in my exosomal samples and in the monocytes. I could not detect any of the other markers in the exosomes, but my positive controls work well so it can't be an antibody problem. However, my proteomics data suggest that all of the mentioned proteins are in my exosome sample. Also, NTA data and electron microscopy suggest presence of exosomes.
What could I do to detect the other markers in my sample?
What method of detection are you using? If you are using fluorescence based methods it may be worth switching to something more sensitive such as ECL due to the low levels of marker protein
Thomas Simon I tried different volumes. Normally I use 40ul from a 200ul stock, isolated from 4ml of human plasma.
Felix Knab you might want to work out the amount of EVs you're working with first... or the amount of proteins. The problem might just be that you're working with very low concentrations of EVs, hence your issues with detecting the markers. In that case, western blotting might not be the ideal method. You might want to look into alternatives such as ELISA.
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