Cell surface ELISA

100 ng of Notch chimera constructs were transfected into U2OS cells in a sterile opaque tissue culture-treated 96-well plate (Corning 353296) in triplicate. 24 hr post-transfection, cells were washed once quickly with 100 uL PBS and fixed using 100 uL 4% PFA (Thermo Fisher 28906) for 20 min at room temperature, then washed quickly three times with 100 uL PBS. Cells were blocked in 100 uL TBS +5% milk at room temperature for 1 hr. Then, 50 uL Flag primary antibody (Sigma-Aldrich F1804) was added 1:250 in TBS +5% milk for 2 hr at room temperature. Cells were washed three times for 5 min each with 100 uL TBS +5% milk at room temperature. The cells were then incubated 50 uL 1:10,000 with an HRP secondary antibody for 1 hr at room temperature before being washed five times for 5 min each with TBS at room temperature. 100 uL chemiluminescent substrate (Dual-Luciferase Reporter Assay (Promega)) was added for 1 min before reading out on a luminescence plate reader.