Protein determination
The Thermo Scientific™ Pierce™ BCA Protein Assay Kit was used to
determined the protein concentrations in each of the exosome samples
obtained using the different isolation methods, IP, TEI, and SEC. Bovine
Serum Albumin (BSA) in 1% SDS was used as a standard with final
concentrations of 2µM, 1µM, 0.5µM, 0.25 µM and 0.125µM. The BCA reagent
(50 ml of solution A and 1 ml of solution B) was added into triplicates
of exosome containing media from different isolation method. Series of
standards and 10 µl of each sample added into a 96-well plate, followed
by 190µl of 1% SDS and 200µl of BCA reagent. The plate was incubated
for 30 minutes, and then read at wavelength of 562nm. BCA Total Protein
Standard Curve is obtained and the protein concentrations were
calculated. The Kruskall-Wallis test is performed to assess whether
there is a statistically significant differences in medians of protein
content in each of the media obtained by different methods.