2.10. Labeling with Activity-Based Probes (ABPs)

Fifty micrograms of protein, extracted from bodies or heads of flies, were incubated with ME569 epoxide (0.5 µM for identification of GCase activity), β-aziridine JJB367 (0.5 µM for identification of GBA1, GBA2, GBA3 activity), TB474 (1 µM for identification of α-galactosidase activity), TB652 (1 µM for identification of β-galactosidase activity), TB482 (3 µM for identification of α-mannosidase activity), TB434 (1 µM for identification of β-mannosidase activity), JJB383 (0.5 µM for identification of α-glucosidase activity) or JJB392 (0.5 µM for identification of β-glucuronidase activity), synthesized at the Department of Bio-Organic Synthesis at Leiden University, as described elsewhere [14,15,16,17,18,19,20], in McIlvaine’s buffer at pH 5.0 (150 mM citric acid−Na2HPO4, pH 5.0) for 30 min at 37 °C. The samples were electrophoresed through 10% SDS-PAGE. Gels were developed by Typhoon FLA 9500 scanner (GE Healthcare, Little Chalfont, UK), capturing Alexa647/Cy5, using PMT 750 V and 100 μm pixel size or by Amersham imager 600 (Amersham, Buckinghamshire, UK).