In Vitro Transcription Translation

[Shaanu]

In Vitro Transcription Translation

• Quote

Post by Shaanu » Mon Jun 03, 2013 7:12 am

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.

[JackBean]

• Quote

Post by JackBean » Wed Jun 05, 2013 12:19 pm

what exactly is autoradiographed if not western-blotted?

[jonmoulton]

Re: In Vitro Transcription Translation

• Quote

Post by jonmoulton » Wed Jun 05, 2013 10:59 pm

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.

[Shaanu]

• Quote

Post by Shaanu » Sat Jun 08, 2013 3:53 pm

Dear Jonmoulton.The pull down certainly gives information on protein protein interaction [as done with the Beads to pull down.] I am not sure whether it made sense or sounds weird to you ::)