I am trying to determine the binding interaction between two proteins. I though of DNA footprinting but my lab is not equipped for radiolabelling, I just need a basic protocol. Any ideas??
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anonymous
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Posted 13 August 2001 - 09:00 PM
Dear Trisha,
Came accross your question on how to proceed with protein-protein interaction. You can study the interaction by two ways :1. In Vitro (CO-IP, Western Pull Down etc..)2. In vivo (Two hybrid systems etc)
I just finished a first part of interaction of CTCF protein with YB 1 as well as DNA Pol. II (holoenzyme), using the 1st approach -in vitro method..
Simply speaking - you need to clone your gene and bacterially expressed. I used pET vector system, with His tag (for further purification)...well, some people use GST system (which is quite straight forward and well established).
Then purify it (the respected region of your protein), bound it to a matrix & use them in Pull Down experiment with He La cells extract. Detection of interaction can be acheived by using the respective antibody.
After you know which region are actually interact, you can proceed with mapping down/search for the actual site - by producing the mutant/truncation.
Good luck.
