I intend to perform footprinting to identify binding site of an RNA-binding protein. All the protocols I read suggest using T4 PNK and [32P]ATP. However, I ordered wrongly [32P]UTP because I am more familiar with labeling in vitro transcription. Is there any other method of RNA labeling using UTP that I can use to do footprinting?
I may be wrong here but I think the T4 PNK can use many other triphosphate nucleotides as phosphate donors to catalyze the phosphorilation reaction, and it should be equally effective. Just make absolutely sure that it's gamma-UTP and you didn't order alpha-UTP by mistake, too. I'm seeing this on ThermoScientific (https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/modifying-enzymes-thermo-scientific/phosphatases-kinases.html), for example. My recommendation would be to carefully read your provider's documentation and see if that's the case for your particular enzyme, or contact them if you don't find information on this.
The best way to go is to use gamma-p32-ATP because of the high energy gamma phosphate bond contributing to the low energy of activation and making the reaction go fast, the gamma phosphate bond of UTP does not have the same energy compared to ATP. If you only have UTP, make sure it is gamma-p32-UTP and increase the reaction time because the efficiency is much lower compared to ATP.
Did you order alpha-32P-UTP or gamm-32P-UTP? If you use UTP for transcription reactions, then you probably ordered alpha-UTP, which cannot be salvaged for T4 PNK labeling because the radioactive phosphate is not at the gamma position.
Carlos is correct. PNK can use gamma-UTP as a phosphor donor
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