Hi Everyone.
I have to find protein and proline content in Barley leaf. Now the question is how to make and find out standard concentrations levels for determining protein and proline in barley leaf ?
Note: As per the procedure, we will then compare the readings from barley leaf with the standard ones.
Thanks in anticipation.
Girija Prasad Patnaik Sir thank you. I am going to use the same procedure as recommended by you. However, i am not clear regarding making the standard solutions of different concentrations and which formula to be used for calculating the standard curve ?? Could you please guide about this. Thanks once again.
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.
Vagmi Singh Vagmi Singh Thanks for your detailed answer. However, how to make the standard curve using different concentrations of L-Proline ?? Could you please answer it. Thanks in anticipation.
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