Protocol
Author
Jennifer Funk
Overview
This assay measures total amino acids. We use it to assess how much N plants allocate to amino acids, but one could also use this method to quantify amino acid uptake and storage.
Materials/Equipment
Equipment:
Mortar and pestle
2ml microcentrifuge tubes
Centrifuge, 4∘ C
Vortex
Bucket of ice
N gas flow for prep of Ninhydrin solution
Plate reader with 570nm filter
96 well plate, new or acid washed
100 ∘C incubator
Chemicals:
Liquid Nitrogen
80% Methanol
Nanopure water (dd H2O)
Sodium Acetate Buffer, pH 5.3, 3.8M with 1mg/100mls NaCN
Ninhydrin solution
Amino Acid standards (Thermo Scientific # 20088)
50% Isopropanol
Procedure
Step 1: Amino acid extraction
Keep everything on ice to prevent protein degradation!!
1. Weigh out 15 to 200mg of frozen leaf tissue. Powder in mortar with pestle with liquid nitrogen.
2. Add 1 ml 80% Methanol and homogenize by grinding.
3. Transfer homogenate to 2ml microcentrifuge tube.
4. Rinse mortar with 1 ml 80% methanol and transfer remaining to 2ml tube.
5. Vortex immediately.
6. Incubate on ice for 20 min.
7. Centrifuge the homogenate for 15 minutes at 10,000g at 4∘ C
8. Transfer supernatant to fresh tube.
9. Store samples at -80∘ C or quantify with Ninhydrin assay immediately.
Amino acid will degrade over time, quantify within 1 week of extraction.
Step 2: Amino Acid quantification (Ninhydrin assay)
10. Amino Acid standard prep: (if standard A is above linear range, delete and use B-F).
| Standard | μl of 5.489 mg/ml Amino Acid std in A | μl 80% methanol | Concentration (μg/ml) | |
| A | 20ul of AA std | 980 | 109.7474 | |
| B | 180 of A | 120 | 65.84843 | |
| C | 180 of B | 120 | 39.50906 | |
| D | 180 of C | 120 | 23.70544 | |
| E | 180 of D | 120 | 14.22326 | |
| F | 180 of E | 120 | 8.533957 | |
| std control | 120 | |||
11. Transfer 120ul of Standard A to new tube for quantification. All standards should have 120ul final volume.
12. Use 2ml microcentrifuge tubes (pre-labeled in step 3)
To each tube add:
120μl amino acid sample, standard, or control
120μl Ninhydrin Solution
Fasten caps.
13. Incubate at 100∘ C for 15 minutes. Sandwich tubes between two microtube racks to prevent caps from opening. Use two C-clamps to secure making sure that the pressure is evenly distributed across the samples.
14. Cool immediately in ice. Leave on ice for 10 minutes.
15. Add 480μl 50% cold (from freezer) isopropanol to each test tube and immediately re-fasten the cap.
If any samples are darker in color than standard A, then dilute 1:2 with more 50% isopropanol. (example: volume of sample + isopropanol is 720ul (240+480), add another 720ul of 50% isopropanol to achieve a 1:2 dilution. If still too dark, remove 500ul of diluted sample, and add it to 500ul of 50% isopropanol in a new tube achieving a 1:4 dilution. Read absorbance of final dilution only.
16. Plate 180μl x 3 replicates from each test tube into a 96 well plate with plate reader ready to read. Read absorbance at 570nm.
Solutions:
1. 5ml Ninhydrin solution (enough for 35 samples)
Add the following to a 15ml centrifuge tube:
.1g Ninhydrin
.015g hydrindantin
Under Nitrogen gas flow add:
3.75 DMSO
1.25ml NaOAC buffer pH 5.3 with NaCN
Bubble with Nitrogen gas for 2 minutes.
Store in refrigerator for up to 8 hours.
2. Sodium Acetate (NaOAC) buffer for Ninhydrin solution, pH 5.3, 3.8M 100mls
(pH 5.3 to 8 ok. Sun et al 2006)
40.34 g Sodium Acetate Trihydrate (403.467g/L)
pH with Glacial acetic acid (50.12g/L, 47.78ml/L)
1 mg NaCN (1mg/100ml, or 2.037ml of .01MNaCN)
up to 100 ml ddH2O
Literature references
Noctor, G; Novitskaya, L; Lea, PJ; Foyer, CH. 2002. Co-ordination of leaf minor amino acid contents in crop species: significance and interpretation. JOURNAL OF EXPERIMENTAL BOTANY 53 (370):939-945
Noctor, G; Bergot, GL; Mauve, C; Thominet, D; Lelarge-Trouverie, C; Prioul, JL. 2007. A comparative study of amino acid measurement in leaf extracts by gas chromatography-time of flight-mass spectrometry and high performance liquid chromatography with fluorescence detection. METABOLOMICS 3 (2):161-174
Sun, SW; Lin, YC; Weng, YM; Chen, MJ. 2006. Efficiency improvements on ninhydrin method for amino acid quantification. JOURNAL OF FOOD COMPOSITION AND ANALYSIS 19 (2-3):112-117