Phos tag aal solution

SDS-PAGE gels (7%) were supplemented with Phos-tag AAL solution (Wako) according to manufacturer’s recommendations. Gels were run at 100V in an XCELL SureLOCK Mini-Cell (Invitrogen) until the dye front completely exited the gel. Gels were incubated in transfer buffer supplemented with 1 mM EDTA for 10 minutes. Gels were then soaked in normal transfer buffer for 10 minutes. Proteins were transferred to a nitrocellulose membrane and standard western blotting procedures were subsequently followed.
For lambda phosphatase treatments, lysates were generated as described in the co-immunoprecipitation assays. 50 μL of lysate was mixed with 10X MnCl₂ buffer and 10X reaction buffer provided with the lambda protein phosphatase kit (NEB). Samples treated with enzyme received 1 μL of purified lambda protein phosphatase. Incubations were performed for 45 minutes at 30°C, and samples were subjected to chloroform-methanol precipitation⁴¹ prior to phos-tag electrophoresis.

Ubiquitin constructs were phosphorylated by incubating 15 µM substrate with PhPINK1 (aa 115-575, concentrations as indicated) in 25 mM Tris (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 10 mM ATP, 1 mM DTT at 22 °C. Reactions were quenched at the indicated time points in EDTA-free LDS sample buffer. Samples were separated with Mn²⁺ Phos-tag SDS-PAGE, using 17.5% (w/v) acrylamide gels supplemented with 50 μM Phos-tag AAL solution (Wako Chemicals) and 50 μM MnCl₂ using an EDTA-free Tris-Glycine running buffer. Gels were stained with Instant Blue SafeStain (Expedeon).