I've isolated exosomes from MCF-7 cells by ultracentrifugation and resuspended the pellet in 1 x PBS. I now need to determine the protein concentration of my exosome preps using a Bradford assay.
My question is, what is the best way to lyse my exosomes to measure protein concentration? I have read that you can use lysis buffers like NP-40 and RIPA, but papers never seem to specify how much lysis buffer to add, how long to add it for, at what temperature, and whether you need to rock or shake the samples while incubating with the lysis buffer.
I'd also love to know if anyone has any better methods for determining the concentration of exosomes? I don't have a nanosight microscope at my institution.
Thank you!
Hi Sabryn!
I lyse my extracellular vesicles with RIPA. I start with a volume of 1 ml of conditioned medium and resuspend in 100 uL of RIPA. And I perform by BCA assay.
Hi Sabryn,
I use micro BCA kit for protein quantification. And I dilute my samples in 2% (v/v) SDS to lyse my EV preparation.
Thank you both for your help!!
Do you use BCA over Bradford for any particular reason? Is it more appropriate for quantifying exosomes (e.g. more sensitive)?
Sabryn Hamila You can`t really use Bradford with RIPA because RIPA contains detergents that interfere with Bradford and give you a blue color as soon as you put Bradford solution in your well (so you don`t get an accurate and correct reading). Which is why BCA is used with RIPA. However you can use Bradford with Pro-Prep!
I usually use 100ul to 200ul of RIPA buffer for protein extraction on my exosome samples.
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