I am tying to detect the interaction between Erk and Importin7 under different stimuli but my results are not so good and the good one did not repeat itself, not even the controls. How can I improve the protocol in these cases?
normally erk-importin 7 interaction is inducible and fairly well described and this interaction is primed by casein kinase 2
Plotnikov A, Chuderland D, Karamansha Y, Livnah O, Seger R. Mol Cell Biol. 2011 Sep;31(17):3515-30
Chuderland D, Konson A, Seger R. Mol Cell. 2008 Sep 26;31(6):850-61
If interaction is weak and transient then alternatively you should try FRET pairs to monitor it in living cells, or may be try a IP with ERK-GOF or in cells with constitutively active MEK.
For short lived protein-protein interactions the stringency of Co-IP experiments may be prohibitive
Co-IP mimic the intracellular conditions for protein-protein interactions and customizing and fine tune the stringency for different protein-protein complexes are therefore important. (Alber et al., (2007) Determining the architectures of macromolecular assemblies Nature 450, 683-694)
You can crosslink protein complexes in your cells briefly with 1-2% PFA prior to lysis. PFA makes very short crosslinks so only closely associated proteins will be stuck together. Then, you don't have to worry so much about your complexes falling apart during washing steps. See this publication: PMID 20634879
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