Hello, my name is Valeria and I am conducting a proline quantification assay following Sunkar 2010 protocol (1). However, upon adding toluene, the red color generated by the ninhydrin-proline reaction fails to migrate to the upper layer where the toluene is located, rendering it impossible for me to quantify the absorbance of my samples. The standards exhibit satisfactory results, indicating no issue with the reagents. The proline-ninhydrin reaction also occurs correctly, as evidenced by the increased color intensity with higher proline concentrations. In an attempt to troubleshoot, I have separately added additional glacial acetic acid and sulfosalicylic acid to samples that did not yield the desired outcome after incubation. However, the red color does not migrate to the upper phase. I am unsure about the cause of the problem in the sample development and would greatly appreciate any assistance. Thank you.
(1) Sunkar, R. (2010). Plant stress tolerance. Methods Mol. Biol, 639, 401.
ISO 690
Hi Valeria
Please make sure that the reaction mixture is carefully and vigorously mixed as the color complex does not migrate by itself, then wait till the mixture is separated into 2 phases; the upper phase contains the toluene and the color complex. More details about your steps would help to explain.
Hi Valeria,
I didn't fully understand your attempt of troubleshooting. Have you tried the assay (with a few samples) without adding ninhydrin? Maybe the color does not derive from a ninhydrin-proline reaction but rather some phytochemicals that are separated by toulene? Maybe your proline concentrations are not very high in your samples and you can still detect it? Proline concentrations can be low depending on a specific stress level or even a plant species. What is the plant species you are working with?
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