Could someone give a stepwise detailed explanation for calculation of proline by Bates et al. method?
Hello Akshatha, here I have shared steps of 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.
Hope this will help you.
Thank you Vagmi Singh ma'am. it would be great if I get an explanation for the calculation
The proline concentration is determined from a standard curve using D-Proline.
In the proline estimation, when converting your absorbance values from the standard curve to µmole/g sample why must I divide the sample weight by 5?
Khansaa A. Hussien , generally the sample is homogenized with 10 ml of 3% sulfosalicylic acid. From the 10 ml sample solution, only 2 ml is taken for the test. Thus the dilution factor is 5. Thus is should be multiplied by 5
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning