I've tried proline determination assay (Bates 1973) on maize leaves, but it didnt work. Though it worked on maize pulverized seed (not lyophilized). Anybody knows if there's something wrong with lyophilized tissue and this specific method? or something with lyophilization procedure?
We always use pulverized or frozen material for proline analysis. It looks from your previous experiment that lyophilized tissue is inappropriate for determination of proline, and if you still want to use lyophilized tissue, I would suggest you to increase the amount of lyophilized tissue for analysis..
Have you tried doing a proline analysis on fresh tissue and with added proline (increasing amounts might be instructive)? Make the same samples then lyophilise them and analyse for proline. that should show if it is lyophilisation which is the problem. P.S. do good replication and perhaps different methods of lyophilisation.
I increased the amount of tissue and seems to be working. Thank you all for your answers.
Andriani Georgios Tzanaki The problem is not lyophilised tissue or its amount. When you remove the frozen material, some of the cytosolutes are wasted outside during the thawing process. Repeated freezing/ thawing process could significantly remove soluble molecules (lets for say proline) and thereby alter with the accurate quantitative determinations. Good luck
Fahad Shafiq the thing is that with the lyophilized tissue i wasn't able to get results. It seems to work with greater amount of tissue
What Kind of material are you using? Were they submitted to any kind of stress before liophization?
Proline determinations:
Proline concentration was determined as described by Bates, Waldren & Teare (1973). Lyophilized tissue (30 mg) was mixed with 1.0 mL of 3% sulfosalicylic acid, sonicated for 15 min at RT and centrifuged for 30 min at 16,000 g. The resulting supernatant was reacted with 3% glacial acetic acid and acid ninhydrin at 95 ◦C for 1 h, cooled to RT, and extracted with 0.8 mL of toluene. After vortexing for 15 s, the toluene layer was removed and the absorbance at 520 nm was measured against a standard curve generated with L-proline solutions of known concentration.
Thank you for your responses. Seems to have worked at about 100-150mg. Tested it at lyophilized beans, the ones that are sold in the market for human consumption (so dont know if they were stressed). And lyophilized maize leaves which werent stressed. 30mg were too low.
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