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What Wavelength Do You Read TMB At?
TMB gives two reliable readings depending on how you run the assay:
| Readout | Condition | What You Measure |
|---|---|---|
| 450 nm | After adding stop solution | The stopped yellow chromophore — most common endpoint |
| 650 nm | Without stopping | The blue reaction product — used for kinetic reading and O.D.-limit checks |
Why 450 nm and 650 nm?
After stopping, the blue product converts to a yellow chromophore whose absorbance peaks near 450 nm — read this for the final endpoint value. Before stopping, the blue form absorbs strongly near 650 nm, which is convenient because it avoids overlap with the detector and lets you monitor color development in real time. Reading at 650 nm without stopping is a common way to run a kinetic (time-course) assay.
Why does TMB change from blue to yellow after stopping?
The blue species is a charge-transfer complex of oxidized TMB. Adding an acid stop solution protonates this complex into a stable yellow diimine, whose absorbance is measured at 450 nm. The absorbance before stopping (650 nm) is proportional to the blue form, while after stopping (450 nm) it measures the yellow form — both correlate with analyte amount, so you can choose either endpoint or kinetic readouts.
Stop Solutions for TMB
To fix the endpoint you can use a dedicated stop solution. Durastab offers TMB Stop Solution 450 and TMB Stop Solution 650 for convenient stopping across different plate formats.
Reading Best Practices
Read within a defined window after stopping to keep results comparable (e.g. read within 30 min).
Subtract blank or read against blank wells for accurate OD values.
Minimize bubbles before reading — they scatter light and inflate absorbance.
Keep temperature consistent between kinetic measurements.
Watch for saturation: if OD exceeds the linear range, shorten development or choose a less sensitive formulation.
Figure 1 | TMB wavelength reading comparison: 450nm vs 650nm
