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I feel like this is a silly question, but if the pellet is invisible, how do you know if you've successfully resuspended it?

Is it best to just pipet gently and wait some length of time before proceeding RNA and protein isolation?  I wish there was something like GlyoBlue that could be added to help with visibility.

On a related note:  the protocol instructions need to be improved for clarity.  The exosome isolation protocol ends with resuspending the pellet in "a convenient volume of 1x PBS or similar buffer", and indicates exosomes are then ready for downstream analysis and are stable for 1 week at 2-8oC or -20oC long-term.  Seems straighforward enough and like a good place to pause and plan the next step.  Except now that I'm looking more closely at the RNA and protein isolation kit, it explicity indicates pellets should be resuspended in ice-cold Exosome Resuspension Buffer if protein analysis is required, and to incubate samples 5-10 min. at RT to dissolve.  (No idea why you'd want ice cold buffer if you need to dissolve at RT.)  Then later it tells you if you're not immediately doing protein to store it 5-10 min. on ice or -20 long-term, or for RNA immediately to -20.  If the choice of buffer and storage timing are that criticial to downstream applications, it should say so in the exosome isolation protocol rather than instruct you to do one thing only to find later you should have done it differently.  The instructions are seemingly contradictory and confusing, which is very frustrating.
asked Aug 12, 2014 in ExosomesTalk by aroff
edited Aug 12, 2014 by aroff

1 Answer

If the pellet is tiny or invisible (which is a typical case when isolating exosomes from small volumes of cell media < 1 mL) it is very easy to dissolve. Pipet it several times up and down, or gently vortex. Larger pellets might require addition of PBS, then you let the tube sit for about 1h, then pipet or vortex. To confirm that exosomes are actually in suspension I'd recommend use of Nanosight LM10 or similar particle counter/sizer.

Regarding the protocols- stability is the key, especially with RNA analysis (with protein its less of an issue). So most of manipulations should be done fast, and ideally at lowest possible temperature. Thus all these precautions in the protocol. Its not deadly if you use room temp buffer, but we just stick to the best practices...
answered Aug 14, 2014 by Vlassosv
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