I am starting from single cell suspension and I am interested in isolating autophagosomes and maybe other vesicles (lysosomes, ER, mitochondria, MVBs). I don't have the instrumentation for nitrogen cavitation and I would like to avoid dounche homogenizer. I have good antibodies for different organelle markers.
Thank you
Hello Matteo,
I think that it is quite challenging to isolate autophagosomes without homogenization of your sample. I found a paper that describes the isolation of LC3-positive membranes (http://www.ncbi.nlm.nih.gov/pubmed/19910472). You won't get intact autophagosomes by following this method but maybe it is a start?
With best regards, Christian
Hi Matteo
I have recently been trying subcellular density fractionation of different vesicles to look at among other things, autophagosomes. I homogenized my samples using either a dounce homogenizer or a "cell cracker"/ball-bearing-device. After homogenization in an isotonic buffer, I would first remove nuclei and other debris by 1000xg for 10 min then sediment my membranes of interest by 70000xg 1h. The pellet was resuspended and added to an opti-prep gradient to separate according to vesicle density and centrifuged 70000xg 16h.
I have been testing many variations of this assay and I do see a good distribution of different vesicle sizes. This has been useful for me to get an idea of how different proteins distribute between vesicle types, and to determine by proteinase protection assay to see if the protein is inside the vesicle or on the outside. However, you will never get fractions that are completely pure of only autophagosomes using this method - then I think you need some sort of affinity capture.
As Christian said I think you need to do some sort of homogenization of your sample, f.ex dounce. The paper he suggested is also good reading and possibly more relevant for your use than what I have been doing. Another paper that I found very interesting while I was reading about and testing different variations of this assay is this one: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736544/
Let me know if you have any questions
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