I have a nuclear protein which can interact with several proteins. I want to do pull down assay to fish a new interacting partner of my protein of interest. But i don't know how much of total mammalian cell lysate (in conc) should I mix with my purified protein to do a success full pull down.
good question I have the same problem, I am going to do this experiment in few weeks. When I obtain results i will tell you.
@ Jakub- Do u have a specific pray protein expressed in a cell line or u want to fish a new protein.................please share ur experience with pull down
How will you fish a interacting partner via pulldown... what will you do after getting the pulled bands in gel.. will u go for peptide sequencing? i am using Y2H to find my partners then i will confirm again in pulldown. How did you made GST library construct?
I have a specific pray protein but I want to check if it is localized in the nucleus. So I use nuclear extraction kit to obtain a nuclear fraction of proteins I will tray to fish these proteins with magnetics beads. After electroforesis and blot transfer i will tray to identify my protein with specific antibodies.
@Kesavan- You can fish a new interacting partner , by using GST tagged bait protein. so u need to mix a total cell lysate with GST- tagged bait protein, followed by column purification. Then run purified stuff in SDS -PAGE and do coomassie staining or silver staining to detect any new band corresponding to new interacting partner.
ajit i have tried this approach to fish my interacting protein partner to a GST purified protein. Firstly, if u have any hint of what probable partner u are looking for then take lysate from a cell line which either do not/or lowly express the protein partner which is already on the GST beads . This is to avaoid any interferance of the endogenous interaction. i have tried with 1mg of total cell lysate to successfully do a pull down. However, if the affinity of two protein interacting is sufficiently high you can even reduce the cell lysate amount. Once i detected my protein partner for further co-ip experiment i had just used 200microgram of the lysate.
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