| Assay Method Information | |
| | TarO Biochemical Enzymatic Assay |
| Description: | The TarO biochemical enzymatic assay is a liquid chromatography-mass spectroscopy (LC-MS) based end point assay that measures C55-P-P-GlcNAc (LIPID III) production. The TarO biochemical enzymatic assay was performed in a 384-well microtiter plate (Labcyte) with a reaction volume of 20 μl. The reaction mix contained 0.1 μgs/μl of TarO membrane preparation derived from MRSA COL (lysostaphin/lysozyme treated, centrifuged at 40K rpm, and re-suspended in 50 mM Tris pH 7.5, 10 mM MgCl2), 1500 μM UDP-GlcNAc, x 75 μM C55-P substrates in 83 mM Tris pH 8.0, 6.7 mM MgCl2, 6 mM CHAPS, and 8.3% DMSO buffer. The enzyme reactions were quenched by extraction in 40 μl of 1-pentanol containing 0.04 μM 15C C55-PP-GlcNAc, which was used as an internal standard. A 10 μl volume of the quenched reaction mixture (pH≈3) from each well was injected onto a reversed-phase column (C4, 5 m, 2.1×50 mm, Thermo Scientific Biobacis-4) and eluted using a NH4Ac/H2O/MeOH gradient (solvent A: 10 mM NH4Ac in water, pH 5.6; solvent B: NH4Ac (1 M)-Isopropanol (1:90, v/v, pH 5.6). The HPLC conditions were as follows: 15% solvent B for 15 seconds followed by a gradient to 90% solvent B in 90 seconds; then solvent B was kept at 95% for 10 seconds followed by a gradient to 8% solvent B in 0.1 minute. The column was then equilibrated at 15% B for 1 minute before the next injection. The flow rate was kept constant at 600 μl/minute. Mass spectrometric detection was carried out in the negative-ion mode using selected reaction monitoring (SRM). Typical mass spectrometric conditions were as follows: heated capillary temperature, 210° C.; spray voltage, 2500 V; desolvation gas (N2), 40 l/h; auxiliary gas (N2), 35 l/h. Selected ion current (SIC) chromatograms of C55-PP-GlcNAc and internal standard 15C C55-PP-GlcNAc were plotted and integrated using LCQuan incorporated in Xcalibur software (ThermoFinnigan). The linearity of C55-PP-GlcNAc concentration versus mass spectrometric signal (AC55-PP-GlcNAc/A15C-C55-PP-GlcNAc) was determined with purified C55-PP-GlcNAc. The IC50 values were calculated using the nonlinear regression analysis (sigmoidal dose response fit allowing for a variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent. |
| Affinity data for this assay | |
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