Hello,
I am trying to purify a specific vesicle, from a fraction extracted from plant cultured cells (P1500g-17000g) using M270 magnetic Dynabeads, but I consistently observe a large amount of an unknown contaminant sticking to my beads after purification process. This contaminant is not related to my target and appears highly autofluorescent, as seen in the image and bright field (red arrow). Has anyone encountered a similar issue or has an idea of what this contaminant could be?
Can you provide information on your imaging protocol? Did you stain your cells with antibody?
There's no staining protocol, the cells I use express a GFP that allows purification with anti-GFP dynabeads. Basically I incubate a membrane fraction with the dynabeads and after the wash steps I put fiew µL of resuspended beads and place them between slide and coverslip. The dynabeads (2.8µm) are the spheres that you can see in BF and are slightly autofluorescent. The images are from Wild Type cells, the contaminant indicated by a red arrow is not related to GFP. In my conditions of interest I capture my GFP-vesicles on the surface of the beads and they are distinguishable from these contaminants.
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