Determining if a protein is a downstream target of another protein - (Oct/23/2008 )
Hi, I have a question as to what methodology can be used to determine this since my background and major is not in molecular/cellular biology. Basically, I was just wondering what methods can be used to determine if the activation of a certain protein is acting downstream of some signaling factor. I assume you can try to block the signaling factor and run a Western for the protein and see if it is present. Is there any other methods of doing this? Sorry if this is not clear.
Thanks.
Thanks.
there are several approaches possible, e.g.: as suggested by Western if you know sth about the signalling mechanism, f.i. phosphorylation/dephosphorylation events; immunoprecipitation studies to show protein protein interaction, phosphorylation and colocalization studies in immuncytochemistry
Hi, thanks for the reply. I'm not too sure what "f.i." phosphorylation is.
Also, is there any good papers that go through the methods.
thanks.
To determine activation of proteins:
- RNAi
- overexpression
- CoIP-pull down
- phosphorylation/dephosphorylation or acetylation or whatever your protein uses to activate the other.
- I agree in mycroscopy to colocalize
- FRET is there is interaction
etc....
Also, is there any good papers that go through the methods.
thanks.
F.i. means for instance: maybe phosphorylation is a topic; you may provide more information to give you sth more concrete...