I'm trying to optimize an ELISA-based technique where I coat with a protein, block, add a second protein and then detect for the second protein.
Do you know any effective protocol?
Also, how reliable is this approach in order to detect protein-protein interactions?
Its fine, if it works.
There are some pitfalls in using ELISA for this. Mostly, protein1 binding to the plastic and antibody binding to protein2 could interfere with the protein1-protein2 binding. The protein-protein binding also might not be strong enough to survive the ELISA washes and handling.
A more direct approach would be observing the complex formation directly, either by gel filtration, native PAGE, etc.
Its fine, if it works.
There are some pitfalls in using ELISA for this. Mostly, protein1 binding to the plastic and antibody binding to protein2 could interfere with the protein1-protein2 binding. The protein-protein binding also might not be strong enough to survive the ELISA washes and handling.
A more direct approach would be observing the complex formation directly, either by gel filtration, native PAGE, etc.
http://jvi.asm.org/content/84/11/5550#ref-list-1
In this report you can see how they determine the affinity between Feline calicivirus capsid protein and its reported cellular receptor protein. the experiment is ELISA based.
Maybe you would have to modify a bit the protocol ( blocking, interaction time) for your particular experiment.
Good luck
Carlos
It works of course but measuring interaction using ELISA can be an hard way. I'm not sure that any effective protocol exists for ELISA but you can find good advices into the litterature. Otherwise, have you considered SPR (Biacore) or ITC to measure your interactions?
Certainly, your approach is not a conventional method to detect protein-protein interaction. Let alone the many pitfalls, drawbacks and artifacts that you will have to deal with. Why don't you consider, co-immuno precipitation, Forster resonance energy transfer, two-hybrid, or as others suggested, biacore methods. But in establishing a new interaction, you will have to use a technique that is broadly accepted.
There is an old but excellent method to quantify antibody affinities in solid phase assays described by Friquet: Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME. J Immunol Methods. 1985 Mar 18;77(2):305-19.
This method starts with the incubation of distinct antigen concentrations with a constant antibody concentration (half saturation) to get the chemical equilibrium adjusted. In the second step the residual antibody will be back-titrated. From the concentrations of the free antibody at the respective antigen concentration the affinity / avidity can be calculated.
This principle will work also for any other protein-protein interaction. On of the proteins has to be at the solid phase. Both proteins has to be incubated as described above. The residual free second protein can bin at the second step to the solid phase protein. The only thin you need is a detection principle for the second protein. A labeled antibody could be the solution.
Using this method affinities from 10E-5 L/mol up to 10E-16 are detectable without an special equipment like plasmon resonance.
Indeed ELISA can be used for checking Protein- Protein interaction. I personally have tried checking interaction between two purified protein, and it came out fine. Later on, I did confirm that binding with immunoprecipitation assay.
It would be really nice if you have antibodies against both the interacting partners. and try using biotin system to amplify signals. and do keep all controls to rule out any non specific binding.
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