Hi everyone,
Do you know a good method to isolate early or late endosomes for cultured cells ?
I tried several protocols with sucrose gradient but i didn't see the interfases. I used 3 T175 per condition.
When I checked on WB, i see a contamination of the cytoplasm in the endosomal fration.
Do you have an other method or maybe some advices ?
Thanks,
Amandine
Hello Amandine.
Even if the best protocol exists, there are many pitfalls in isolation of the endosomes. Approriate cell lysis, temperature of the sample, quick working practice without breaking, selection of the gradient matrice, proper wash of the fractionated endosomes, use of inhibitors, and finally adapting your different cells in the chosen protocol. Please study the wonderfully prepared presentation that has all critical points.
https://www.i-med.ac.at/cellbio/info/vorlesungen/vorlesung_wien_huber-0805.pdf
Best wishes.
Thank you very much Sang Ho,
it is a very nice presentation and thanks for the advices !
Best wishes,
Amandine
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