New liquid biopsy method offers potential for non-invasive Parkinson'sdisease testing LifeAtPurdue NaturePortfolio
. In brief, the frozen urine samples were thawed in a 37 °C water bath. The samples were then centrifuged at 2500 ×for 10 min to remove cell debris and large apoptotic bodies and diluted with EVtrap loading buffer in a 1:10 v/v ratio. The magnetic EVtrap beads were added directly to the diluted at a ratio of 20 µL EVtrap beads per 1 mL urine.
The Western blot experiments were carried out to show biological replicates rather than technical replicates . Therefore, all of the Western blot experiments were performed only once due to a very limited amount of rare clinical samples. A small percentage of each purified EV sample was incubated for 10 min at 95 °C in LDS sample buffer. The equivalent volume of each sample aliquot was loaded and separated on an SDS-PAGE gel .
For the validation experiments, the membranes were cut according to the appropriate molecular weights to detect the target proteins at their corresponding molecular weights before blocking and incubated with the following primary antibodies: rabbit anti-CD9 at 1:5000 ratio, or rabbit anti-STK11 at 1:1000 ratio, or mouse anti-PCSK1N at 1:1000 ratio, together with rabbit anti-HNRNPA1 at 1:1000 ratio.
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