Nativepage cathode buffer additive

Blue native‐PAGE was performed as indicated by the manufacturer. Protein extracts and immunoprecipitation (IP) eluates were added with 4× NativePAGE™ Sample Buffer (Invitrogen™, BN2003) and NativePAGE™ 5% G‐250 Sample Additive (Invitrogen™, BN2004) to a final concentration of 0.125%. Then, samples were loaded on NativePAGE™ Novex® 3–12% Bis‐Tris Gels (Invitrogen™, BN1001) and run at 150 V in cathode buffer containing Coomassie G‐250 (by adding NativePAGE™ Cathode Buffer Additive to 1/200 dilution, Invitrogen™, BN2002 to NativePAGE™ Running Buffer, Invitrogen™, BN2001). NativeMark™ Unstained Protein Standard (Invitrogen™, LC0725) or SERVA Native Marker (SERVA, 39219.01) were loaded to predict the size of detected protein species.

BN-PAGE was performed as previously described [75 (link), 76 (link)]. Mitochondria (150 μg) were solubilized in 50 μl of NativePAGE Sample Buffer (4X; Invitrogen, catalog # BN20032) containing digitonin (Invitrogen, catalog # BN2006) at a digitonin/protein ratio of 4 g/g, on ice for 20 min. Solubilized proteins were supplemented with Coomassie brilliant blue G-250 (Invitrogen, catalog # BN2004) at a ratio of digitonin/Coomassie dye of 4:1. Samples were separated on 3–12% NativePAGE Bis-Tris gels (Invitrogen, catalog # BN2012BX10) and run with dark blue cathode buffer at 30 V for 1 h. The cathode buffer was then replaced with light blue buffer and the gels were run for 20 h at 30 V at 4°C. Dark blue cathode buffer was prepared by adding 50 mL of 20X NativePAGE Running Buffer (Invitrogen, catalog # BN2001) and 50 mL of 20X NativePAGE Cathode Buffer Additive (Invitrogen, catalog # BN2002) in 900 mL of deionized H₂O. Light blue cathode buffer was prepared by adding 50 mL of 20X NativePAGE Running Buffer and 5 mL of 20X NativePAGE Cathode Buffer Additive in 945 mL of deionized H₂O.

The mitochondrial pellet was resuspended in 100 μl of PBS, and 5 μl was taken for protein quantification. Next, Medium A was removed by centrifuging at 10,000 x g for 5 minutes, and the pellet containing the isolated mitochondria was solubilised and processed as previously described for EVs and NSCs.
A total of 50 μg of protein for each of the samples was loaded into a pre-cast NativePAGE 3–12% Bis-Tris gel (Invitrogen). For the run, 1x NativePAGE Running Buffer plus 1x NativePAGE Cathode Buffer Additive (Invitrogen) was added to the cathode, and 1x NativePAGE Running Buffer was added to the anode. Halfway through the run, the cathode buffer was substituted for 1x NativePAGE Running Buffer plus 0.1x NativePAGE Cathode Buffer Additive and run until the front reached the end of the gel.

Samples diluted with 4X sample buffer (50 mM BisTris-HCl, pH 7.2, 50 mM NaCl, 10% w/v glycerol, and 0.001% Ponceau S) were loaded onto a NativePAGE 3%–12% Bis-Tris Gel (Invitrogen) and run in the cold room at 4°C. The cathode buffer was 50 mM Tricine, 50 mM BisTris-HCl, pH 6.8 plus 1X NativePAGE Cathode Buffer Additive (Invitrogen) and the anode buffer was 50mM Tricine, 50 mM BisTris-HCl, pH 6.8. Gels were run at 150 V for ∼60 min and then the voltage was increased to 250 V for the remainder of the run ∼90 min.