Application

This kit can be used to measure the Proline (Pro) content in plant tissue and honey samples.

Detection significance

Proline is an proteinogenic amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated NH2+ form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −COO− form under biological conditions), and a side chain pyrrolidine, classifying it as a nonpolar (at physiological pH), aliphatic amino acid. It is non-essential in humans, meaning the body can synthesize it from the non-essential amino acid L-glutamate. Proline is the only proteinogenic amino acid with a secondary amine, in that the alpha-amino group is attached directly to the side chain, making the α carbon a direct substituent of the side chain.

Detection principle

Proline can react with acidic-ninhydrin to form stable red compound. The maximum absorption peak of the compound is at 520 nm. And the concentration of Pro can be calculated by measuring the OD value at 520 nm.

Experimental instrument

Micropipettor, Vortex mixer, Water bath, Centrifuge, Spectrophotometer (520 nm).

Sample pretreatment

Honey sample: Weigh 0.02-1 g of honey to the tube, add Extraction solution at a ratio of tissue (g) : Extraction solution = 1: 10. Incubate in 100℃ water bath for 15 min (shake the tube constantly), cool to room temperature, centrifuge at 10000 g for 15 min, collect the supernatant for detection.

10% tissue homogenate: Weigh 0.02-1 g of fresh tissue and wash with double distilled water for several times, then absorb with filter paper and weigh accurately, add Extraction solution at a ratio of tissue (g) : Extraction solution = 1: 10. Homogenized mechanically with a homogenizer. Then centrifuge at 10000 g for 15 min, collect the supernatant for detection.

[Note]: If the sample can’t be tested on the day, it can be stored at -80℃ for a month.

Operation steps

Mix fully and incubate in 100℃ water bath for 30 min, then cool with running water. Add 4 mL of methylbenzene (self-prepared), oscillate fully and stand for 10 min. Take the supernatant to another centrifuge tube, centrifuge at 2325 g for 10 min and collect upper red methylbenzene layer. Set to zero with methylbenzene and measure the OD value of each tube at 520 nm with 1 cm optical path cuvette.

Technical parameter

1.  The sensitivity of the kit is 0.17 μg/mL.

2.  The intra-assay CV is 4.5% and the inter-assay CV is 5.4%.

3.  The recovery of the kit is 105%.

4.  The detection range of the kit is 0.17-25 μg/mL.

Notes

1. This kit is for research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 6 months.

4. Do not use components from different batches of kit.

5. Please operate in fuming hood. Methylbenzene is poisonous, please wear mask and glove.

6. If the concentration of proline in samples is out of the detection range of the kit, please dilute the samples appropriately before the assay and multiply the dilution factor in calculation formula.