I doubt that the coloured solution will interfere at 240nm. Regardless, I have blanked the medium.
Simple test then. Blank at 240nm with 1mL of your medium, add 1uL of 30% H2O2, mix. Check Abs240nm. Repeat. See if you see stepwise increase in 240nm. My guess is that in a test tube setting with no cells or proteins, this test will work just fine.
The problem with this method, is that many cell culture medium components such as amino acids will also absorb at 240nm. If you see an absorbance change at 240nm, you don't know if it's caused by H2O2 or any other component in the medium. Also, the free transition metals (iron, copper, etc) would quickly react with H2O2. I wouldn't recommend this method.
there are fluo sensors of ROS or H2O2. check Mol Probes (now Invitrogene). at 240 nm, i think no chance to measure specific signal in any kind of TC medium.
thank you guyz.
yes I do know we have methods to measure H202.
i can titrate with KMnO4. or use 240nm.
bt can i detect H2O2 just at 240nm in any colored solution where it will not react with the components of colored solution.
r the color of the solution will interfere at 240nm.
@ Siyuan Su if i remove phenol red the medium becomes colorless then no point of my question.
I just want to verify can we measure UV range compounds in colored solutions.
Simple test then. Blank at 240nm with 1mL of your medium, add 1uL of 30% H2O2, mix. Check Abs240nm. Repeat. See if you see stepwise increase in 240nm. My guess is that in a test tube setting with no cells or proteins, this test will work just fine.
The problem with this method, is that many cell culture medium components such as amino acids will also absorb at 240nm. If you see an absorbance change at 240nm, you don't know if it's caused by H2O2 or any other component in the medium. Also, the free transition metals (iron, copper, etc) would quickly react with H2O2. I wouldn't recommend this method.
Gian Chuang, do you know were I can read more about interference at 240nm? Because I'm having problems with catalase quantification.
Thanks!
Challagundla, sorry for using your topic!
This answer is kind of late but might be still of value for you and others. You can use Hyperblu from Lumigen to directly measure H2O2 in your medium. This generates chemiluminescence, which can be measured in plate reader. The advantage is that you do not need an enzyme and additional substrate to generate a colorimetric reaction. Medium and Hyperblu are simply mixed in a well
This is a recent paper using this technology:
Food Chem. 2015 Feb 1;168:183-9. doi: 10.1016/j.foodchem.2014.07.050 [Titel anhand dieser DOI in Citavi-Projekt übernehmen] . Epub 2014 Jul 14.
Critical assessment of the formation of hydrogen peroxide in dough by fermenting yeast cells.
Rezaei MN1, Dornez E1, Verstrepen KJ2, Courtin CM3.
To Monya:
as far as I understood it, you can also use HyperBlu to indirectly measure the activity of enzymes metabolizing H2O2.
Best regards,
Christian
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