Green Color Upon Adding Stop Solution (Streptavidin-HRP) (ELISA (R&D Guide))

Question

Accepted Answer

When sulfuric acid stop solution is added to wells after TMB substrate incubation, a green color appears instead of the expected yellow, indicating incomplete mixing and pH gradient in the well. Common causes: 1) Reagents not adequately mixed in wells after stop solution addition 2) Insufficient tapping or agitation of plate after adding stop solution Solutions: 1) Gently tap the side of the plate 5-10 times after adding stop solution to ensure thorough mixing 2) Visually confirm uniform yellow color in all wells before reading plate 3) If green color persists, briefly agitate plate on orbital shaker at low speed (200 rpm, 10 seconds)