I am using Luminol based chemiluminescence for detection of ROS in Arabidopsis leaves and seedlings. Blank (Luminol+HRP+KOH, without any tissue), is generating luminescence higher than the positive control (Arabidopsis seedlings + 10uM Flg22 + Luminol + HRP + KOH). I am sure that none of the reagents are contaminated. But still I get readings in Blank. Is the Blank supposed to give such high readings? The attached graph shows luminescence values for blank, untreated and treated arabidopsis seedlings with Flg22
Why in your protocol are using KOH? Probably this -OH is reacting with luminol.
Dear Neha Kachewar,
My friend is also encountering the same sort of problem and I am writing on her behalf.
She used LUMINOL from Biorad. Blank used was (Luminol+HRP+Water, without any tissue). Still there is high RLU values in the blank compared to other groups. The RLU values decreases with passes of time.
BTW, which is the LUMINOL supplier in your case?
Not sure if it helps your problem, but I experienced that the activity of the peroxidase matters for the amount of background, you can test a range of Peroxidase amounts or a fresh batch and see if it solves your problem.. It does not explain however why your blanks are having a higher signal than the tue experiments. Looking at your graphs the ROS response is very fast. Doesn't is generally come up only after 5-10 minuites?
Moriconi Jorge I.
Thank you for your suggestion.
I am using 0.2M KOH because the luminol powder does not dissolve in water. I had referred this paper for the assay: Jeffrey C. Anderson et. al. Arabidopsis MAP Kinase Phosphatase 1 (AtMKP1) negatively regulates MPK6-mediated PAMP responses and resistance against bacteria (2011) The Plant Journal, 67, 258–268. In the paper the authors have mentioned that the Luminol stock (500X) was prepared in 0.2M KOH. I had followed this. My stock and working solutions: both are in 0.2M KOH. I think I should use water instead of 0.2M KOH to prepare working solution from stock. This will reduce the effective -OH conc. in the final solution.
Kshitiz Raj Shrestha
Is your friend using KOH while preparing Luminol stock and working solutions?
I have used Luminol from Calbiochem and Sigma. But in both cases I have encountered the same problem: High RLU values in Blank
Mark Kwaaitaal
I agree with you. Whatever papers I have seen/referred for ROS, Flg22 induced ROS RLU values start increasing after 5-10 mins and then peak. In my case, I don't know why the response is that fast.
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