Far-Western Blotting
Studying Protein Interactions by Far-Western Blotting
Far-Western blotting was originally developed to screen protein expression libraries with 32P-labeled glutathione S-transferase (GST)-fusion protein. Far-Western blotting is now used to identify protein:protein interactions. In recent years, far-Western blotting has been used to determine receptor:ligand interactions and to screen libraries for interacting proteins. With this method of analysis it is possible to study the effect of post-translational modifications on protein:protein interactions, examine interaction sequences using synthetic peptides as probes, and identify protein:protein interactions without using antigen-specific antibodies.
Far-Western Blotting vs. Western Blotting
The far-Western blotting technique is quite similar to Western blotting. In a Western blot, an antibody is used to detect the corresponding antigen on a membrane. In a classical far-Western analysis, a labeled or antibody-detectable “bait” protein is used to probe and detect the target “prey” protein on the membrane. The sample (usually a lysate) containing the unknown prey protein is separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) or native PAGE and then transferred to a membrane. When attached to the surface of the membrane, the prey protein becomes accessible to probing. After transfer, the membrane is blocked and then probed with a known bait protein, which usually is applied in pure form. Following reaction of the bait protein with the prey protein, a detection system specific for the bait protein is used to identify the corresponding band (Table 4).
| Table 4. Comparison of Western Blotting and far-Western Blotting Methods | ||
| Step | Western Blotting | Far-Western Analysis |
| Gel Electrophoresis | Native or Denaturing (usually) | Native (usually) or Denaturing |
| Transfer System | Optimal membrane and transfer system determined empirically | Optimal membrane and transfer system determined empirically |
| Blocking Buffer | Optimal blocking system determined empirically | Optimal blocking system determined empirically |
| Detection (several possible strategies)* | Unlabeled primary antibody.–>Enzyme-labeled secondary antibody.–>Substrate Reagent | Unlabeled bait protein.–>Enzyme-labeled bait-specific antibody.–>Substrate Reagent |
| Enzyme-labeled primary antibody.–>Substrate Reagent | Radiolabeled bait protein.–>Exposure to film | |
| [Arrows designate sequence of steps of detection strategy] | Biotinylated antibody.–>Enzyme-labeled streptavidin.–>Substrate Reagent | Biotinylated bait protein.–>Enzyme-labeled streptavidin.–>Substrate Reagent |
| Fusion-tagged bait protein.–>Tag-specific antibody.–>Enzyme-labeled secondary antibody.–>Substrate Reagent |
*Labeled antibodies generally are enzyme-labeled (either horseradish peroxidase or alkaline phosphatase). By contrast, bait proteins generally are not enzyme-labeled since a large enzyme label is likely to sterically hinder unknown binding sites between bait and prey proteins. Other labeling and detection schemes are possible.
Specialized far-Western Analysis
By creative design of bait protein variants and other controls, the far-Western blotting method can be adapted to yield very specific information about protein:protein interactions. For example, Burgess, et al. used a modified far-Western blotting approach to determine sites of contact among subunits of a multi-subunit complex. By an “ordered fragment ladder” far-Western analysis, they were able to identify the interaction domains of E. coli RNA polymerase ß subunit. The protein was expressed as a polyhistidine-tagged fusion, then partially cleaved and purified using a Ni2+-chelate affinity column. The polyhistidine-tagged fragments were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The fragment-localized interaction domain was identified using a 32P-labeled protein probe.
Importance of Native Prey Protein Structure in far-Western Analysis
Far-Western blotting procedures must be performed with care and attention to preserving as much as possible the native conformation and interaction conditions for the proteins under study. Denatured proteins may not be able to interact, resulting in a failure to identify an interaction. Alternatively, proteins presented in non-native conformations may interact in novel, artificial ways, resulting in “false positive” interactions. The prey protein in particular is subjected to preparative processing steps for far-Western blotting that can have significant effects on detection of protein:protein interactions. This is not to imply that identification of valid interactions is not possible but only to stress the importance of appropriate validation and use of controls.
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