0.05 g ground, frozen leaf tissue was extracted in 5 ml of 3 % sulfosalicylic acid (Panreac, Barcelona, Spain) by sonication for 30 min. After centrifugation at 4000 g for 20 min at 4 °C, extracts were assayed for proline as described by Bates and others [31 (link)] with slight modifications. Briefly, 1 ml of the supernatant was mixed with 1 ml of glacial acetic acid and ninhydrin reagent (Panreac) in a 1:1 (v:v) ratio. The reaction mixture was incubated in a water bath at 100 °C for 1 h. After centrifuging at 2000 g for 5 min at 4 °C, absorbance was read at 520 nm. A standard curve was performed with standard proline (Sigma-Aldrich, St. Louis, MO, USA).
Ninhydrin reagent
Manufactured by ITW Reagents
Ninhydrin reagent is a chemical compound used in forensic science and biochemistry. It is primarily used for the detection and visualization of amino acids, peptides, and proteins. The reagent reacts with these compounds to produce a distinctive purple-blue color, known as the 'Ruhemann's purple' color reaction. This reaction is a sensitive and reliable method for the identification and analysis of various biological samples.
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3 protocols using ninhydrin reagent
1
Proline Quantification in Leaf Tissue
2
Leaf Proline Quantification Protocol
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Proline analysis was performed as described by Bates et al. (1973) (link) with some modifications. Briefly, 50 mg of ground leaf tissue was extracted in 5 ml of 3% sulfosalicylic acid (Panreac, Barcelona, Spain) by sonication for 30 min. After centrifuging at 4000 × g for 20 min at 4°C, 1 ml of the supernatant was mixed with 1 ml of glacial acetic acid and ninhydrin reagent (Panreac) in a 1:1 (v:v) ratio. The reaction mixture was incubated in a water bath at 100°C for 1 h and subsequently centrifuged at 2000 × g for 5 min at 4°C. Finally, absorbance was read at 520 nm. A standard curve was assayed with pure proline (Sigma-Aldrich, St. Louis, MO, United States).
3
Proline Quantification in Frozen Leaves
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We performed proline analysis as described by Bates et al. (1973) with some modifications. We extracted 50 mg of ground frozen leaf tissue by sonication for 30 min in 5 ml of 3% sulfosalicylic acid (Panreac, Barcelona, Spain). After centrifuging at 4000×g at 4 °C for 20 min, the supernatant was mixed with glacial acetic acid and ninhydrin reagent (Panreac) in a 1:1:1 proportion (v:v:v). We incubated the reaction mixture at 100 °C for 1 h. After centrifugation at 2000×g at 4 °C for 5 min, proline concentration was spectrophotometrically determined at 520 nm. We performed a standard curve with pure proline (Sigma-Aldrich, St. Louis, MO, USA).
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