Far-WesternBlot is a molecular biological method, which is based on the technique of Westernblot, to detect in vitro protein-protein interactions, especially for detecting interactions that do not require the native structure of the protein of interest.
The overall workflow of far-Western blot technique is similar to Western blotting, except that antibodies are needed for probing target proteins in Western blotting. Whereas in far-Western analysis, antibodies are not needed, instead a purified and labeled "bait" proteins are used to probe and detect a target "prey" protein on the membrane. In both Western blotting and far-Western blot, proteins are separated either SDS gel of native PAGE gel, followed by transferring proteins from the gel onto the membrane. After transfer, the membrane is blocked with BSA with empirical concentration. Then proteins on the membrane are denatured and renatured, which is an optional step and depends on the binding conditions needed for the experiments. The membrane is then probed with a known highly purified bait protein. Following the reaction of the bait with the prey protein, a detection system, dependent upon the bait protein used, signals of band that corresponds to the prey protein can be detected.
Critical factors of Far-Western Blot Analysis
Though Far-Western blot is a convenient way and has been widely used to study protein-protein interaction, cautions must be taken when preserving the native conformation and interaction conditions for the proteins. There are a few problems regarding far-Western blot. First, for proteins separated in a SDS gel, which is a denaturing condition, proteins are denatured. It is possible that denatured proteins may not interact with bait proteins, resulting in a failure to identify an interaction. Secondly, proteins are denatured and renature before binding assay, which may also leads to a potential conformational change of the protein, affecting the binding affinity of target proteins to the bait protein. Thirdly, if the exposure of binding motif is regulated by other factors, there can be a chance that the binding motif is buried inside and become unreachable to the bait, and cause a failure of detection of interaction. What’s more tricky is that, proteins presented in non-native conformations may interact in novel, artificial ways, resulting in false-positive interactions. Creative Proteomics can design the experiment based on your needs, and assess the potential factors that may affect results. We promise reliable data with high reproducibility.
Features of Far-Western Blot Analysis
Workflow of Far-Western Blot Analysis
Our Far-Western Blot methods contain: