I want to measure protein and nucleic acid within conditioned medium. I used nanodrop with fresh medium as blank. Yet, I got a weird result, the blank absorbance is higher than the sample. However, I expected that the blank will have lower absorbance since it doesn't contain growth factors or other molecules released by cells. If I use PBS or ddH2O as blank, is it comparable? since, I used medium containing phenol red. Thank you
You can't use media for nucleic acid (NA) measurement! I am not sure what you are measuring in conditioned medium without purifying the NA first. NA are measured at UV range (260nm) and proteins at 280nm, while phenol red has a reading at 560nm. Proteins of various types can be found in conditioned medium, but if you use medium without serum or any additives (growth factors, antibiotics) just basic medium it may serve as blank for proteins. You see in fresh medium the unused serum (FBS) proteins and compounds will give high reading, whereas in conditioned medium it has been mostly metabolized by cells. Still i don't see a value of measuring conditioned medium directly! It's best to do parallel Trizol isolation of RNA, DNA and proteins from the medium then measure them separately. As for using ddh2o or pbs as blanks is wrong, a blank should be the same solution/solvent you used to suspend your target molecules of interest.
Hello
Not only nucleic acids & protein absorb UV light, so it seems like that your media contain some compound that absorb at 260 &280nm, theerfore it gave you high reading.
you can use the protein concentration using one of the colorimetric methods and so is the case with nucleic acids so that you will be more specific and reduce the interference that you have with the UV method.
Good luck
The medium containing phenol red has an influence on the detection of light signal, especially fluorescence.We detected the secreted protein in the medium using phenol-red medium, and the corresponding blank should also be phenol-red medium, not water or PBS.
For reference only , I hope it can help you .
good luck!
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