For protein extraction, 50 mg of fresh plant material were extracted in the extraction buffer (50 mM TRIS–HCl, 0.5 mM MnCl2, pH 8). One millilitre of the extraction buffer was added to each sample and was centrifuged for 20 min at 10,000 g, 4°C. The supernatant was recovered and stored at −20°C. For LAP activity, Leu‐p‐nitroanilide (Sigma) was prepared from the stock solution as enzyme substrate at a concentration of 3 mM in a solution of 50 mM TRIS‐MnHCl2. The stock solution was previously prepared at 150 mM in ethanol and stored at −20°C. To carry out the analysis, 40 μL of the protein sample and 200 μL of the substrate were incubated for 15 min at 37°C and absorbance was measured at 410 nm as described in (Chao, Pautot, Holzer, & Walling, 2000 ). β‐1,3‐glucanase activity was measured by the Somogy‐Nelson method as described by Román et al. (2011 ).
Leu p nitroanilide
Manufactured by Merck Group
Leu-p-nitroanilide is a synthetic substrate used in laboratory settings to detect and measure the activity of the enzyme leucine aminopeptidase. It is a colorimetric reagent that produces a yellow color upon cleavage by the enzyme, allowing for quantitative analysis.
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2 protocols using leu p nitroanilide
1
Plant Protein Extraction and Enzymatic Assays
2
Plant Protein Extraction and Enzyme Assays
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For protein extraction, fifty milligrams of fresh plant material were extracted in the extraction buffer (50 mM TRIS-HCl, 0.5 mM MnCl 2 , pH 8). One mL of the extraction buffer was added to each sample and were centrifuge for 20 minutes at 10,000 g , 4ºC. The supernatant was recovered and stored at -20ºC. For LAP activity, Leu-p-nitroanilide (Sigma) was prepared from the stock solution as enzyme substrate at a concentration of 3 mM in a solution of 50 mM TRIS-MnHCl 2 . The stock solution was previously prepared at 150 mM in ethanol and stored at -20ºC. To carry out the analysis, 40 μL of the protein sample and 200 μL of the substrate were incubated for 15 minutes at 37ºC and absorbance was measured at 410 nm as described in (Chao et al., 2000) (link). β-1,3 glucanase activity was measured by the Somogy-Nelson method as described by Roman and co-workers (de Roman et al., 2011) (link).
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