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How to deal with delta absorbance readings resulting in negative values?

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#1 djw23



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Posted 17 February 2018 - 06:11 AM

Hi there,


I have a specific problem I was hoping someone could help me with.  I’ve purchased a kit that measures Total Antioxidant Status at an absorbance (abs) of 660nm.  I plan to use this kit to measure TAS in tissue lysates where I use the same protein concentration for each sample so it is standardised between samples and plates.

The protocol involves adding assay buffer to samples, water/lysis buffer (as neg control) and a standard (as positive control, 1mM Trolox).  The plate is then read at 660nm to obtain a blank reading.  Then ABTS solution is added to each well and the plate is left at either 37°C for 5 minutes or 10 minutes at room temperature.  Antioxidants in the sample reduce dark blue-green colored ABTS radical to colorless reduced ABTS form. A second reading is then obtained at 660nm and the second abs is then subtracted from the first abs for each sample/control.  The change of absorbance at 660 nm is related with total antioxidant level of the sample. 

TAS is then calculated as : 

(Δabs water/lyis buffer  - Δabs sample)


(Δabs water/lysis buffer – Δabs standard)


The problem I’m having is that Δabs for water or lysis buffer is lower than the Δabs of my samples so I end up with negative values when solving the top line of the above equation.  I also don’t see much of colour change between adding the ABTS and taking the second reading although this is only to the naked eye, the data suggest a reaction is occurring.  I’ve tried using different volumes of reagent:sample and I’ve still been getting these negative values, although it seems to be only when I incubate the plate at room temp for 10 minutes. I haven’t tested the possibility that both my water and lysis buffer may be contaminated so I will test that next.  I’ve run maybe 3-4 assays and 2 out of those throw these negative values for when I subtract Δabs sample from the Δabs for water/lysis buffer.


I’d really appreciate if someone could advise on why they think I’m getting these negative values from the assay and how my values for water and lysis buffer are lower than my samples as I’m currently baffled as to why this is happening!  I’d greatly appreciate any response as I have only have a finite amount of reagents left in the kits for me to complete my experiment.


I have attached a xcel sheet with my data for you to look at if interested.


Many thanks in advance,





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Edited by djw23, 17 February 2018 - 07:20 AM.

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