Green Color Upon Adding Stop Solution (Streptavidin-HRP)

Symptom

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.

1. 1 Reagents not adequately mixed in wells after stop solution addition
2. 2 Insufficient tapping or agitation of plate after adding stop solution

1. 1 Gently tap the side of the plate 5-10 times after adding stop solution to ensure thorough mixing
2. 2 Visually confirm uniform yellow color in all wells before reading plate
3. 3 If green color persists, briefly agitate plate on orbital shaker at low speed (200 rpm, 10 seconds)

Related Video (3)

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"Official R&D Systems Quantikine hands-on guide matches the R&D ELISA protocol context and demonstrates the stop/substrate steps where the green-color failure occurs."

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How to Run an R&D Systems Quantikine ELISA

"R&D Systems Quantikine walkthrough explicitly includes troubleshooting guidance, directly relevant to diagnosing incomplete mixing/pH gradient after stop solution."

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"Invritrogen step-by-step kit tutorial demonstrates proper TMB substrate and stop solution handling, useful for learning correct mixing technique."