Assay for cytoplasmic ENGase

 Culture cells of your interest. Collect cells.

 Perform all the subsequent procedures either on ice or at 4^oC. Suspend cells at a density of 5 x 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 x complete^TM protease inhibitor cocktail (Roche)/1 mM AEBSF (Pefabloc SC, Roche)).

 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 x g for 1 hour.

 Soluble (cytosol) fraction thus obtained can be used for ENGase assay. For later use, the sample can be aliquoted, stored at -80^oC. 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^oC for 6 hours (incubation time has to be optimized for your enzyme source).

 Stop the reaction by heating sample at 95^oC 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^oC.

 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; 7.5x 75 mm; Tosoh Corp.)

Column temperature: 30^oC

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.