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Membrane fractions prepared from transiently transfected HEK293 cells were solubilised in 1% Triton X-100 and 0.1% SDS in phosphate buffered saline (PBS; 150 mM NaCl, 2.6 mM KCl, 10 mM Na₂PO₄, 1.9 mM KH₂PO_4, pH 7.4). Samples were centrifuged (21,255×g, 15 min, 4 °C) to remove debris. Endoglycosidase H (EndoH, 5 mU/100 µl of sample) or endoglycasidase F (EndoF; 1 U/100 µl of sample; Roche Diagnostics GMBH, Mannheim, Germany) were added to equal aliquots of solubilised membrane samples and incubated overnight at 37 °C [40 (link)] before immunoblot analysis.

With the purpose of distinguishing between glycoproteins that have not reached the mid-Golgi and folded, processed, mature glycoproteins, cell lysates containing 30 µg of total protein were subjected to overnight incubation at 37 °C with 5 µL of Endo-H or 2.5 µL of Endo-F (Roche, Basel, Switzerland) upon denaturing the samples for 5 min. at 95 °C in Endo-H or Endo-F denaturing buffer (Endo-H Buffer: buffer sodium citrate 50 mM pH 5.2, PMSF 0.5 mM, SDS 0.1%, Triton X 100 0.5%, mercaptoethanol 0.1 M; Endo-F Buffer: Buffer sodium phosphate 100 mM pH 7.2, EDTA 10 mM, Triton X 100 0.1%, SDS 0.1%, mercaptoethanol 1%). Digested and undigested samples were then separated on a 4%–20% gradient mini-protean TGX pre-cast gel (BioRad, Hercules, CA, USA) and western blot analysis was performed using anti-GBA 2E2 antibody, as described above.