I want to study whether two proteins: Myc-tagged protein A and Flag-tagged protein B, interacts with each other or not. For that I co-expressed these proteins in HEK293T cells, and later (after 48 h) I lysed the cells and subjected to co-immunoprecipitation analysis.
I found no interaction between these two proteins.
I just have a thought that some additional signal might require to induce modification of one protein, and that results in binding.
Sure, stimulation via specific molecules (depending on your targets) may induce interaction of both.
What are yiur tagged potentially interacting proteins/factors?
Hi Sailesh,
You raised a nice question. However, before I answer that, I would like to tell that I am no technical expert in Protein-Protein interaction analyses and I am going to answer it through my theoretical knowledge.
Firstly, since you used correct procedure and did not find any interaction, therefore it is technically correct to say that there is no interaction between those proteins. You conclude whatever you find through your experiment, anything apart from that are subject to further evaluation.
There might be some additional molecule that might be able to induce structural modification in any of the protein, resulting in the binding. But it is not that easy to discover such novel interaction (if not reported in the literature). Another way to understand any possible interaction is through simulated bioinformatics. Therefore you can consult with a bioinformatics colleague if you think that there might be an additional signal for binding.
Additionally, have a look at the following points which might help:
- Covalent modifications
- Ligands concentrations: Specific substrates or ions can be important factors for protein binding.
- Presence of other molecules: Try to reduce the presence of other proteins, nucleic acids, and ions these as much as possible, which might interfere the interaction
- Protein affinity for proteins or other binding ligands.
- Protein concentration.
- Electric fields around proteins.
Hope this helps a bit.
- Badges
- Science method