Amino Acid extraction and quantification



Jennifer Funk


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.



Mortar and pestle

2ml microcentrifuge tubes

Centrifuge, 4 C


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


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


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.


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


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