Hello,

following a modified protocol of Proline quantification by Shabnam et al. 2016, Bačkor et al. (2004) and Lee et al. (2019), I tried to quantify proline in probes and also to establish a calibration curve. The concentrations from 0.01-1mM went fine, but from higher concentrations (above 10mM) began to precipitate out of solution (see picture), in the standard solutions as well as the probes. My questions are :

1) Is this high concentration of proline causing the precipitation? 20mM is quite a bit and likely not relevant, I just wanted a very wide range for our own notes.

2) Is there something wrong with the protocol I am using below?

Extraction

- Filter biomass onto filters

- Extract in 1mL 1% v/v sulfosalicylic acid @ 70C for 1 hour, then overnight at 4C

Quantification

- Preheat heat oven to 100C

- Centrifuge eppis @ 13K for 5 min

- 0.5 mL algae extract/standard + 1mL ninhydrin solution (ninhydrin 1.25% w/v in 80% glacial acetic acid)

- Incubate at 100C for 60 min in drying oven

- Stop reaction on ice for 10 min

- Transfer to cuvettes and read at 512 nm/ ninhydrin solution as "blank"