Why measurement under 630nm is too low around 0.7（Pi is 10nm in the well）, whether the color of solution is normal ?
Is it necessary to add Oxalic acid?
Malachite green reagent 1.2% malachite green in 1.5M sulfuric acid : 8% Ammonium molybdate tetrahydrate in ddH20:Tween20 (100:25:2)
The Pi standard solution is in 100μL (from 0μM to 100μM ）in every well add 25μL Malachite Green reagent for 5min then add 10μL 5% oxalic acid. Finally measure the optical density of each well at 630nm.
and the standard curve of phosphate not very beautiful?
[The curve in attachments that x axis is Pi concentration(μM) and y axis is measurement OD630nm]
Thank you for your attention！
I've used Malachite Green/molybdate-based reagents with a sensitivity down to about 2 µM phosphate. One such method is described here:
Article A High-Throughput Absorbance-Based Assay for Methionine Prod...
This method does not have a stabilizer added after the MG reagent, so you must be consistent about the reading time.
The method described here includes a stabilizer and is also pretty sensitive, if I remember correctly.
https://www.sciencedirect.com/science/article/abs/pii/0003269788900024
When making the reagent, it is important to avoid adding any contaminants that could interfere. Laboratory glassware or plasticware is sometimes contaminated with phosphate-based detergent residue. Wash it thoroughly with distilled, deionized water.
Adam is suggesting that you may have a problem with reaction conditions to which I would add perhaps pipetting
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