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Preparation of Urinary Exosomes by Nanomembrane Concentrator
Nanomembrane concentrators (Vivaspin 500, $3 for each 500 ul of tube; Vivaspin 4, $4 for
each 4 ml of tube or Vivaspin 20, $8 for 20 ml of each tube; Sartorius Inc., Goettingen,
Germany) were washed to remove glycerol and other preservatives by adding approximately
one volume of PBS buffer then centrifugation at 3,000 × g at room temperature (RT) before
processing urine samples. After centrifugation of urine samples at 17,000 × g at 4 ºC for 15
min, 0.5 ml, 1.5 ml or 10 ml of urine supernatant was added to a Vivaspin 500, 4, or 20,
respectively, nanomembrane concentrator and then centrifuged at 3,000 × g at 20°C for 10–
30 min. The urine proteins were recovered from the nanomembrane concentrator by two
methods: 1) A combined retentate (CR) was recovered by adding an equal volume of
unheated 2X solublizing buffer to the retentate while still in the concentrator and shaking at
RT for 30 min, or 2) sequentially recovering the proteins not bound to the nanomembrane
and adhering proteins: the retentate (R) was removed from the concentrator before adding an
equal volume of the pre-heated (95°C) 2X solublizing buffer (2X Laemmli buffer with 400
mM dithiotheritol (DTT)); the nanomembrane was subsequently washed to remove
remaining proteins that adhered to the nanomembrane by adding two volumes of pre-heated
1X solublizing buffer (HSW) into the concentrator and shaking the concentrator at RT for
10 min (Fig. 1). Urine exosomal proteins isolated by ultracentrifugation (200,000 × g
pellets) described previously(7) were used as positive controls. The combined retentate (CR)
method was used to isolate from the urine of 9 patients (male=5, female=4) with FSGS and
8 normal control volunteers (male=4, female=4). All of the urinary protein samples were
stored at −80°C until use.
https://www.ncbi.nlm.nih.gov/pmc ... /pdf/nihms42388.pdf |
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|Archiver|手机版|外泌体之家 | exosomes & microvesicles
发表于 2017-8-2 13:06:13
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