| Assay Method Information | |
| | Cell-based ELISA |
| Description: | The ELISA portion of the assay is performed in a black Maxisorp 96-well plate that is coated overnight at 4° C. with 100 uL/well of the cell lysate (1:10 dilution of the lysate with PBS containing protease inhibitors, phosphatase inhibitors, and PMSF). The following day the wells are washed 3 times with 300 uL/well of Wash buffer (Tris-buffered saline with 0.1% Tween 20). The wells are blocked with 100 uL/well Blocking buffer (Tris buffered saline w/0.05% Tween 20 and 2.5% Bovine serum albumin). Each well is then washed two times with 300 uL/well of wash buffer. The anti O-GlcNAc antibody RL-2 (Abcam, Cambridge, Mass.), diluted 1:1000 in blocking buffer, is added at 100 uL/well. The plate is sealed and incubated at 37° C. for 2 h with gentle shaking. The wells are then washed 3-times with 300 uL/well wash buffer. To detect the amount of RL-2 bound horse-radish peroxidase (HRP) conjugated goat anti-mouse secondary antibody (diluted 1:3000 in blocking buffer) is added at 100 uL/well. The plate is incubated for 60 min at 37° C. with gentle shaking. Each well is then washed 3-times with 300 uL/well wash buffer. The detection reagent is added, 100 uL/well of Amplex Ultra RED reagent (prepared by adding 30 ul of 10 mM Amplex Ultra Red stock solution to 10 mL PBS with 18 uL 3% hydrogen peroxide, H2O2). The detection reaction is incubated for 15 minutes at room temperature and then read with excitation at 530 nm and emission at 590 nm. |
| Affinity data for this assay | |
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