Assay for cytoplasmic ENGase

 Culture cells of your interest. Collect cells.

 Perform all the subsequent procedures either on ice or at 4°C. Suspend cells at a density of 5 × 10⁷ cells/mL in 10 mM Tris-HCl buffer (pH 7.5)/1 mM EDTA/250 mM sucrose/1 mM DTT with various protease inhibitors (1 × complete^TM protease inhibitor cocktail/1 mM AEBSF (Pefabloc SC)).

 Homogenize cells using Potter-Elvehjem homogenizer or equivalent.

 Clear the solution first with regular centrifuge at 14,000 rpm for 10 min

 Take supernatant to a new tube.

 Centrifuge the sup further using ultracentrifuge at 100,000 × g for 1 h.

 Soluble (cytosol) fraction thus obtained can be used for ENGase assay. For later use, the sample can be aliquoted, stored at −80°C. Avoid repeated freeze-thawing.

 Mix 1 mL of the cytosol fraction with 3 μL of 200 mM Mes-NaOH buffer (pH 6.0) and start the reaction by adding 1 μL of the substrate (Man₉GlcNAc₂-PA; 2 pmol) to the solution.

 Incubate at 37°C for 6 h (incubation time has to be optimized for your enzyme source).

 Stop the reaction by heating sample at 95°C for 5 min.

 Add 1.5 volume of ethanol (final concentration; 60%) to the sample, and centrifuge at 14,000 rpm for 15 min at 4°C.

 Take supernatant to a new tube.

 Evaporate the sup to dryness using centrifugal evaporator.

 Dissolve the sample with 5 mL of distilled water for HPLC analysis.

 Prepare buffer A (0.1 M ammonium acetate buffer, pH 4.0) and buffer B (0.1 M ammonium acetate buffer, pH 4.0/0.5 % n-butanol).

 Elution conditions are as follows:

Column: ODS column (TSK gel ODS-80-TM; 7.5 × 75 mm; Tosoh Corp., Tokyo, Japan)

Column temperature: 30°C

Flow rate: 1.0 mL/min

Elution condition: linear gradient of 5% (0.025% n-butanol) to 100% (0.5% n-butanol) buffer B for 55 min. Wash the column with the starting buffer (5% B solution) between the analyses.

Fluorescence detection: λ_ex=320 nm; λ_ex=400 nm.