I am trying to estimate the proline level of tobacco seedlings under normal and NaCl treatment in Wt and transgenic tobacco lines. In normal conditions the level of proline in tobacco seedling was estimated by using Bates protocol and after calculating the value with the formula given in bates protocol I am getting the lowest value of 23 ug/mg fresh weight. But when I was checking some article. I found that the value of proline is ranging from 1 to 5 ug/mg fresh weight.
Your concentrations (not 'levels') seem high, but you need to check them against several published results - use Google. Be careful with units and wet versus dry weight basis.
If you confirm your values are high, I recommend you check your lab dilutions, and then check your calculations (from first principles, not a recipe). If you still think your values are high, I recommend you re-do the experiment. And make sure you are personally in-charge, not a laboratory technician.
I also recommend you think critically about why you are measuring proline at all. I suggest you re-examine your research question. You might keep your current research question, but your research overall will be strengthened from this critical reflection. I encourage you to do this because the role of proline in adaptation to abiotic stress has come and gone from fashion in research since the 1980's, but still the role is not clear (at least when I last looked). For example, read the review:
M. Ashraf and M. R. Foodlad, “Roles of Glycine Betaine and Proline in Improving Plant Abiotic Stress Resistance,” Environmental and Experimental Botany, Vol. 59,
2007, pp. 206-216.
Similarly, see Pospilova et al 2011 who concluded, using tobacco, that "... the clear correlation be-tween proline content and stress tolerance was not proved in our experiments." (in American Journal of Plant Sciences, 2011, 2, 318-324)
You might like to see simplest and eco-friendly protocol for proline quantification that appeared online in Protoplasma (you may download the same from my ResearchGate site).
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