I have a co-stimulator A on CD4 T cell membrane. D is a transcription factor, responsible for CD4 differentiation. According to the known signalling pathway graph, perhaps there are two protein B & C, during the signalling pathway between A and D. What kind of technique can detect the indirect protein interaction between A and D? Mass spectrometry?
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If the proteins form a complex, co-immune precipitation is the usual technique: Incubate the sample with an antibody against one protein, precipitate the antibody-protein complex (for example with Pansorbin, dead, protein A expressing Staph. aureus cells) and then perform electrophoresis and detect which proteins are also in the complex. You could perform a Western for another protein, if you have the suspicion that it is in the complex. The more general approach is to metabolically label the sample (extract it from cells grown in the presence of 35S-Met/Cys) and then perform an autoradiography.
Another approach would be FRET: the proteins suspected of interacting are labelled with fluorescent dyes. The acceptor dye has an excitation maximum identical to emission maximum of the donor dye. Thus, if the labels are close together (in a complex), energy is resonance-transferred from the donor to the acceptor dye, the intensity of fluorescence at the wavelength of the donor dye decreases, and emission at the maximum of the acceptor dye increases. The effect depends on the 6th power of the distance, which can be calculated with high precision.
If the proteins are merely on the same pathway, but do not interact directly, the analysis is more complicated and may involve switching off the expression of a particular protein (for example with siRNA or a double-minus mutant). If that protein was essential, the pathway should no longer work.
The gene coexpression concept is an indirect method to detect the protein interaction, but it must be used as a complementary approach in order to validate interactions generated from biochemical methods.
Some variants of affinity purification (AP-MS) are well suited for characterizing indirect interactions and partners of the same network. Check for instance proximity labeling method.
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