Ninhydrin Reagent

Acetic acid 80 ml
ddH2O 12 ml
Conc. H3PO4(85%) 8 ml
Ninhydrin 1.25 g

Mix until dissolved

Assay:

1. Ground tissue with 5ml of 3% 5’-sulfosalicylic acid (in water).
2. Mix 1 ml Ninhydrin reagent with 0.5 ml sample and boil 1 hr
3. Extract color with 1 ml toleune 
4. Measure OD at 520 nm
5. Standard curve could be obtained by adding pure proline at 1 to 100 nmoles
    to each assay)

Reference:  Bates L. S., R.P. Waldren, and I.D. Teare (1973) Rapid
determination of free proline for water stress studies. Plant and Soil 39:
205-207