I am trying to carry out experiments which will prove the interaction between two different proteins. One protein is present in the cytoplasm and is thought to be secreted into the environment, and the other protein is a transmembrane protein with the N terminal in the extracellular space and the C terminal coming into the cytoplasm. I have tried bacterial two hybrid experiments but didn't seem to see any interaction. However if the protein is acting on the transmembrane protein at the extracellular component then i won't be able to detect that using two hybrid experiments. What are the easiest ways to detect interaction of these two proteins using molecular microbiology techniques or cell biology techniques?
The easiest way will depend on how much information you already have about these two proteins, and the techniques that you are familiar with or are commonly used in your lab.
Is the structure of the two proteins known? The extracellular domain of the transmembrane protein is what you need - not the whole molecule. If so, you can try Molecular Dynamics simulation or protein-protein docking to see the interaction between the two proteins in silico. This will not provide proof though, you will still need to do in vitro experiments.
Are you able to purify the proteins? Again, just the soluble domain is all you need. You could tag each protein with a pair of fluorophores and use FRET (fluorescence resonance energy transfer) to detect interaction. Or you could detect the binding without any labeling, using ITC (isothermal titration calorimetry). You might even be able to detect binding by monitoring internal fluorescence of the proteins if there is such a signal as well as significant perturbation upon interaction.
How strong is the interaction expected to be? If you expect it to be very strong, you might be able to see it by running the proteins on a size exclusion chromatography column or even gel electrophoresis (non-denaturing). If the proteins bind to each other strongly, a mixture of the two will elute as a much larger molecule.
Hopefully you find one of these methods easy enough. This is not meant to be an exhaustive list, so you might find something else that works better for you.
Good luck!
The easiest way will depend on how much information you already have about these two proteins, and the techniques that you are familiar with or are commonly used in your lab.
Is the structure of the two proteins known? The extracellular domain of the transmembrane protein is what you need - not the whole molecule. If so, you can try Molecular Dynamics simulation or protein-protein docking to see the interaction between the two proteins in silico. This will not provide proof though, you will still need to do in vitro experiments.
Are you able to purify the proteins? Again, just the soluble domain is all you need. You could tag each protein with a pair of fluorophores and use FRET (fluorescence resonance energy transfer) to detect interaction. Or you could detect the binding without any labeling, using ITC (isothermal titration calorimetry). You might even be able to detect binding by monitoring internal fluorescence of the proteins if there is such a signal as well as significant perturbation upon interaction.
How strong is the interaction expected to be? If you expect it to be very strong, you might be able to see it by running the proteins on a size exclusion chromatography column or even gel electrophoresis (non-denaturing). If the proteins bind to each other strongly, a mixture of the two will elute as a much larger molecule.
Hopefully you find one of these methods easy enough. This is not meant to be an exhaustive list, so you might find something else that works better for you.
Good luck!
You could try to use a chemical crosslinker to make a covalent linkage between the two proteins in the cellular environment, then use an antibody (or a tag) to co-immunoprecipitate them and Western blot to detect them.
Hi Jess, if you have an antibody to the transmembrane protein, you could try immunoprecipitation (IP).
I would recommend MST (MicroScale Thermophoresis). It's an easy, fast and precise way to quantify biomolecular interactions. You could express the transmembrane protein as a fluorescent fusion protein (as the authors did in the attached paper) and just add the other protein in a dilution series. With the fusion protein, you could do the measurement even in cell lysate without the need to purify the fusion protein.
Hello Heidi, this is interesting technique (MST) that I was not aware of. Do you need specialized equipment to monitor the signal? If so, how much does it cost?
Hi Nitesh,
thanks for your interest. The Monolith series instruments offer MicroScale Thermophoresis. There are 4 different instruments available with different specifications.
Please feel free to contact me directly for further information.
http://www.nanotemper-technologies.com/products/monolith-series/
Hi Jess,
one possibility is to fused your proteins of interest with fragment of the Infra-red fluorescent protein for protein-fragments complementation assay (IFP-PCA). These fragment were design to be innert in normal condition, but recover the fluorescence when proteins of interest are close in space (less than 8nm). You could therefore fused your transmembrane protein on the intracellular and the extracellular portion to determine if your soluble protein interact inside or outside the cell.
Here is the article for the IFP-PCA:
An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions.
Nature Methods 11, 641–644 (2014).
Good luck
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