I have been working for months on trying to get a Co-Immunoprecipitation to work between proteins X and Y and have been unable to do so thus far. I am thinking of switching to ELISA but have no prior ELISA knowledge or experience. Can anyone recommend any links or protocols that I can use as a jumping off point for protein-protein interaction through ELISA?
Hi Charles,
First you should optimize the ELISA. optimized the concentrations of reactants you use. Then you can carry out the assay. please find the attached optimization protocol and handbook on ELISA. they will guide you to do this.
There are three main methods in ELISA. sandwich, indirect and direct ELISA. first of all you should have to determine which method you are going to use. then you can optimize the assay.
it's not very clear what you are going to analyze? can you brief how you are going to analyze? are the proteins are purified or crude protein?
optimization protocol: http://www.fitzgerald-fii.com/media/Protocols/ELISA_Optimization_Protocol.pdf
I recommend for studies looking at protein-protein interaction to use Bia-Core.
Hi Charles, if your IPs are not working, it could be that your Ab is blocking the interactions between protein X and Y. Are you using a polyclonal or monoclonal for the IP?
I would highly recommend MicroScale Thermophoresis (MST). It's an easy, fast and precise method to quantify biomolecular interactions. This technique measures the motion of molecules along microscopic temperature gradients and detects changes in their size, charge or hydration shell.
The great advantage for you would be to measure free in solution, without any immobilization to surfaces. There are also no Abs required, which could interfere with the binding site. If you express one of the proteins as a fluorescent fusion protein, you could measure this interaction directly in cell lysate without any purification step.
Following this link, you will find a lot of publications where MST has been applied to investigate protein-protein interactions:
http://www.nanotemper-technologies.com/applications/biomolecular-interactions/protein-protein/
You can also contact me directly if you have any question on how to perform the assay using MST.
Hi,
I agree with Vinit that YFP protein-fragment complementation assay (PCA) could be another option. However, this assay is irreversible and would prevent you from determining a Kd. Another option would be to used the IFP PCA which is fully reversible.
PMID: 24747815
It has been test both in vitro and in vivo.
Best of luck!
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