Question (75891) | 650 nm Stop Solution for TMB Substrate (ab171531)

The lab that provides the 650nm TMB stop solution to us sent data that shows no difference in detection limit compared to the standard 450nm stop solution. The 650nm actually had the advantage of increased dynamic range. In their words: "The yellow product has a higher extinction coefficient that yields about 2.6 times the absorbance signal for a given concentration of analyte. There is an ongoing perception that converting the TMB to the yellow product increases the ability to detect the analyte... Addition of the yellow stop clearly produced higher signals at each analyte concentration thereby showing an increased sensitivity; however, some points were saturated at an OD ≥ 4.0. A benefit of reading at 650 nm is that these points now fall in the quantifiable range....The increase in standard deviation for each point is about 2.6-fold higher for the 450 nm reading versus the 650 nm reading. This is equivalent to the signal increase achieved by stopping and reading at 450 nm. So, the signal and the error have been increased by exactly the same factor. This results in no net change in the detection limit....There was no difference in the detection limit or the ability to distinguish between msIgG concentrations across this experiment. Stopping the TMB reaction in the blue and reading at 650 nm is a valuable strategy to increase the dynamic range of an assay without sacrificing the detection."