Good Morning, My name is Maria and I'm working at NYU....

protocol Protocol: RNA-protein UV-crosslinking Assay
Good Morning, My name is Maria and I'm working at NYU. I'm reading this protocol and I have a few questions about UV-crosslinking Assay. I'd like to try it but mixing protein + ATP-32P: 1- You're using a-32P UTP and I have to use g-32P ATP, Is that a Read more Read less more less
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Partho Sarothi Ray Author Answered Nov 8, 2017

Department of Biological Sciences, Indian Institute of Science Education and Research, India

Hello Maria, 1. You cannot use gamma-P32-ATP for this experiment. This is because for UV-crosslinking you require "body-labeled" RNA, i.e. RNA in which the P32 is incorporated within the RNA molecule. Gamma P32 isotopes are used for end-labeling, where only the 5'-phosphate of the RNA is radiolabeled. Such a probe cannot be used for UV-crosslinking experiments as here the RNA is digested after binding with the protein. 2. The UV crosslinking cannot be done in eppendorf tubes, but has to be done in 96-well plates (any make). This is because the reaction as to be exposed to the UV at a short distance , either in an UV crosslinker or using an UV torch, which cannot be done using eppendorf tubes. 3. UV exposure time of 10 min is usually enough for most UV-crosslinking experiments. Only if you do not obtain a RNA-protein complex after 10 min of UV exposure, you should try to increase the time. I hope these answers your queries. Best wishes
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