Hi, I would like to know how I can purify phagosomes from the rest of the cell organelles. I will infect cells with bacteria; once they are wrapped in the phagosome where they will reside, I would like to purify it. I need to keep the membranes intact. Can anyone suggest me a protocol?
Hi Cristián, are you looking to isolate lysosomes?
https://www.piercenet.com/instructions/2161845.pdf
Dear Steingrimur,
Thanks for the advice. For this particular project, I’m trying to purify the phagosomes, the vesicles containing the pathogen (in this case, bacteria). This phagosome sometimes fuses with lysosomes and/or late endosomes, etc., but is not a lysosome.
Anyway, the protocol is very useful for me. Thanks again.
Hi Cristián, I did not realize that the phagosomes were distinct from lysosomes/endosomes and clathrin coated vesicles..
Anyway, here are some papers that I found on isolating phagosomes. I hope it helps.
http://www.pnas.org/content/105/44/16952.full
http://www.biomedcentral.com/1471-2121/9/35
http://jcs.biologists.org/content/122/4/499.full
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1694822/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC443261/pdf/jcinvest00053-0352.pdf
Are the bacteria permanent residents of the phagosomes? If so, you may want to look at Coxiella papers; if not it is going be very time sensitive, as most bacteria that transiently reside in phagosomes escape quite quickly (< 30 minutes). Even so, the Coxiella and Rickettsia fields may have protocols that could be helpful. You will need a method of differential centrifugation. Good luck
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