Hi Radhika,

To be able to study the interaction of other proteins between these two proteins, you can carry out the following experiment:

1. Do you have antibodies against one or both proteins? If you do, I suggest an immunoprecipitation in an environment which has the proteins naturally present. Eg., if the proteins are of human origin, use a cell lysate from human cells.

2. Simple immunoblot of the immunoaffinity pulldown with the second protein will confirm that the 2 proteins are indeed interacting with each other.

3. If you can also immobilize the antibody and repeat the pulldown, you can start to analyze and characterize the identity and relative amounts of the interactome using mass spectrometry based proteomics.

4. In the final data, you should both of the proteins plus others, in varying relative concentrations.

Good luck,

Hediye.

Affinity capillary electrophoresis is a good technique to investigate the interaction between any two molecules; inorganic and organic: small or large molecules such as proteins). The interaction investigation will be based on a change on the mobility of the target molecule after interaction with the ligand. Visit my researchgate and read my thesis which explains simply how can be use ACE technique for binding studies.

With the availability of the purified proteins, you can consider various biophysical approaches, depending on what equipment is available, including surface plasmon resonance and isothermal titration calorimetry. Chemical crosslinking can also be a useful technique.

Hi Radhika,

Here are some suggestions:

• Quantitative methods in vitro: Fluorescence spectrometry, Surface Plasmon resonance (SPR) and Isothermal Titration Calorimetry (ITC)
• Structural methods: NMR spectrometry, X-ray crystallography and Cryo-EM

I recommend you an article I read before, maybe can help you choose the experimental method for determining the structure (https://www.creative-biostructure.com/comparison-of-crystallography-nmr-and-em_6.htm).

Hi Radhika,

as you are working in Würzburg, you could access many great biophysical tools to study protein interaction quantitatively in vitro, such as Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), Fluorescence spectrometry, surface plasmon resonance (SPR), biolayer interferometry (BLI). Proteins that have been purified for crystallography can easily be subjected to these assays.