Determination of Proline (Bates et al. 1973)
1. Plant Dry matter(0.05 g) is homogenized in 5 ml of 3% aqueous sulfosalicylic acid.(can be determined in fresh tissue as well (0.25g) in 10 ml of 3% aqueous sulfosalicylic acid).
2. Leave for 3hrs for extraction to complete
3. Centrifuge at 1500 g for l0 min.
4. 2 ml of supernatant is added to 2ml Glacial Acetic acid and 2 ml Acidic Ninhydrin.
(Warm 1.25 g ninhydrin in 30 ml of GAA + 20 ml (6M) H3PO4 with agitation until dissolved)
5. Boil at 100oC in a Water Bath for 60 min.
6. Stop the reaction by placing in an ice path.
7. Add 4 ml of Toluene and mix vigorously.
8. Let warm to r. t.
9. Read at 520 nm, using toluene as blank.
10. At 4 0C, the reagent is stable for 24 h.
11. Use a standard curve for concentration from 0- 512 µL (20-100 µg/ml) of L-Proline.
The analysis is non specific with regard to plant species or crop.
Go to- www.plantstress.com/methods -and look for 'Proine analysis' down the list.
Please follow these researchgate links
https://www.researchgate.net/publication/43132146_Methods_for_Determination_of_Proline_in_Plants
https://www.researchgate.net/publication/211353600_PROTOCOL_Extraction_and_determination_of_proline
Article Methods for Determination of Proline in Plants
Article PROTOCOL: Extraction and determination of proline
Yes Suresh I agree this is standard procedure for proline estimation.
I also used spectrophotometry as described by Abraham et al 2010. Link is posted by Arvinda Singh above. Easy, accurate and requires less chemicals and reagents.
Determination of Proline (Bates et al. 1973) is the standard method which can be followed. The protocol given by Suresh naidu is correct you can follow it.
Bates et al 1973 is the standard method for proline estimation andvcan bevuse for any plant tissue
Determination of Proline (Bates et al. 1973) is the standard method to be followed
It is possible to determine the content of proline from dry matter
Determination of Proline 1. Plant Dry matter fresh tissue as well (0.5g) in 10 ml of 3% aqueous sulfosalicylic acid). 2. Leave for 3hrs for extraction to complete 3. Centrifuge at 1500 g for l0 min. 4. 2 ml of supernatant is added to 2ml Glacial Acetic acid and 2 ml Acidic Ninhydrin. (Warm 1.25 g ninhydrin in 30 ml of GAA + 20 ml (6M) H3PO4 with agitation until dissolved) 5. Boil at 100oC in a Water Bath for 60 min. 6. Stop the reaction by placing in an ice path. 7. Add 4 ml of Toluene and mix vigorously. 8. Let warm to r. t. 9. Read at 520 nm, using toluene as blank. 10. At 4 0C, the reagent is stable for 24 h. 11. Use a standard curve for concentration from 0- 512 µL (20-100 µg/ml) of L-Proline.
Please also take a look at the following RG link and PDF attachments.
Article PROTOCOL: Extraction and determination of proline
Protocol: Extraction and Determination of Proline (Bates et al. 1973)
1. Plant Dry matter(0.3 g) is homogenized in 8 ml of 3% aqueous sulfosalicylic acid.(can be determined in fresh tissue as well (0.25g) in 10 ml of 3% aqueous sulfosalicylic acid).
2. Leave for 3hrs for extraction to complete
3. Centrifuge at 1500 g for l0 min.
4. 2 ml of supernatant is added to 2ml Glacial Acetic acid and 2 ml Acidic Ninhydrin.
(Warm 1.25 g ninhydrin in 30 ml of GAA + 20 ml (6M) H3PO4 with agitation until dissolved)
5. Boil at 100oC in a Water Bath for 60 min.
6. Stop the reaction by placing in an ice path.
7. Add 4 ml of Toluene and mix vigorously.
8. Let warm to r. t.
9. Read at 520 nm, using toluene as blank.
10. At 4 0C, the reagent is stable for 24 h.
11. Use a standard curve for concentration from 0- 512 µL (20-100 µg/ml) of L-Proline.
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