😀 Hi . I have recently started working with in vitro linked transcriotion:translation to prove my in vivo results of interactions
between two proteins. I used rabbit reticulocytes system and analysed the pull down on commassie gel with S-35 already added in reaction and autoradiographed to visulalize.. Do i have to also prove by western blot ? There are no background lysate proteins shown up in gel. Its pretty good. Any advice. I want to save time and money both as mine is not a funded project.
I will appreciate your help.
It sounds like the autoradiography is done directly on the gel. Since the protein contains the hot sulfur, you don’t need a radiolabeled antibody to detect it. You can infer the mass of the protein from its mobility (location of gel band). However, if you need to unambiguously prove the identity of the protein you might need a Western as well, since your radiolabelled gel gives mass information but not an epitope-antigen interaction. If you have a single mRNA produced in the transcription-translation system then that simplifies the system. Without knowing the source of the DNA and how many different kinds of mRNA would be expected to be present, it’s tough to tell whether a Western would be necessary to prove your band is what it should be.