ELISA Method

Method Coat ELISA plate with 50ul/well 0.2-10ug/ml protein (purity should be above 3%) in PBS or carbonate buffer for 2h on R/T or O/N in the fridge Wash 3x with PBS-Tween (alternative: following washing incubate in ELISA-blocking buffer for 30min R/T and wash 3x with PBS-Tween) Incubate in PBS-Tween diluted antibody/protein solution for 1h at 37C (ideally after 5h reach the equibrium) Wash 3x with PBS-Tween Develope with TMB Add 100ul TMB developing solution to wells For 1 ELISA plate mix: 11ml TMB buffer 110ul TMB solution 22ul H2O2 {Hydrogen peroxide 30% solution Sigma H-1009} Add 100ul 2M H2SO4 to stop the reaction Detect absorbance at 450nm