Journal: BMC Nephrology

Article Title: Strategy and rationale for urine collection protocols employed in the NEPTUNE study

doi: 10.1186/s12882-015-0185-3

Figure Lengend Snippet: Western blot of exosome pellets. Exosomal proteins of interest including podocalyxin (PODXL), fibrocystin (FIBRO) and polycystin 1 (PC1) were examined by Western blot after storage and compared with freshly-isolated exosomes. These proteins were best preserved by the Sigma protease cocktail. a Polycystin 1 detection by Western blot was stable for 1 week at room temperature after exosomes were isolated and stored in 0.01 % sodium azide ( n = 7). However, polycystin 1 was not detected well from exosomes isolated after urine was frozen.at -80C probably because of precipitation issues and unlikely due to loss of protease activity. Effects of preservatives on Western blot of exosome pellet proteins PODXL, scramblase (PLSCR1), FIBRO, and smoothened (SMO). b Lane 1, Fresh, no preservatives; Lane 2, Fresh, no preservatives; Lane 3, −80 °C Frozen/Thawed; Lane 4, −80 °C Frozen/Thawed. c Comparison of varied amounts of protease inhibitors on exosome protein detection by Western blot. Initial assessment and following 6 months storage at −80 °C. Lane 1, 4.8 μL. Sigma protease inhibitor; Lane 2, 4.0 μL Sigma protease inhibitor; Lane 3, 1:100 Roche Complete tablet; Lane 4, −80 °C Frozen/Thawed; Lane 5, Fresh, no preservatives. d Exosomal Podocalyxin fared well at RT and at 4 °C. Sodium azide did not affect their survival. e Analysis of exosomes extracted from frozen (−80 °C) raw urine stored for 12 months

Article Snippet: Scramblase (PLSCR1) was well preserved with Sigma protease inhibitor and one freeze/thaw cycle.

Techniques: Western Blot, Isolation, Activity Assay, Protease Inhibitor