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What is Far-Western Blot (Far-WB)?
The Far-WB is an advanced technique derived from Western blot (WB), which proves to be valuable in investigating direct protein-protein interactions (PPI) in vitro, delineating protein domains or motifs involved in interactions, and identifying potential ligands or interacting partners for a given protein. The Far-WB technique differs from WB in that it employs a labeled bait protein, which is a highly purified protein or protein mixtures, as a probe to specifically identify binding partners of the prey protein on membrane. The bait-prey proteins complex formed after incubation, can readily be identified through methods such as immunodetection or fluorescent labeling. This technique allows researchers to investigate the intricate molecular mechanisms underlying diverse biological processes, thereby providing invaluable insights into protein function, and signaling pathways, ultimately facilitating a comprehensive understanding of complex protein networks.
The Feature of Far-WB
The investigation of PPI commonly employs sophisticated techniques such as immunoprecipitation (IP), Co-IP, and pull-down assays. However, the far-WB is very differed from those methods. Because it usually directly binds to denatured and separated proteins immobilized on a membrane, the method has an advantage in detecting interactions within short, linear peptide motifs. Nevertheless, detecting interactions that rely on the native, folded conformation of the target protein may pose challenges or even prove unfeasible for Far-WB. Therefore, it is often used to confirm direct interaction following IP, Co-IP, and pull-down assays as well.
Our Far-WB Analysis Service
The Far-WB is routinely used for confirmation of the known PPI and discovery PPI between known and unknown proteins. Here is a process for performing a Far-WB in Creative Proteomics.
1) Experiment design: The optimal steps for each stage are determined based on the sample type and the characteristics of the specific proteins under investigation. We could also help to select appropriate positive and negative controls to enhance the interpretation and explanation of the results.
2) Protein extraction: Use appropriate lysis buffers to extract the prey proteins of interest from cells or tissues. If necessary, prey protein purification can be further performed.
3) Native PAGE: Proteins are usually separated by either SDS-PAGE or native PAGE, with native-PAGE preferred for preserving protein's native conformations as much as possible.
4) Protein transfer to a membrane: Reproduce the prey protein onto a NC or PVDF membrane like the WB procedure.
5) Blocking: Incubate the membrane with skim milk or bovine serum albumin buffer.
6) Incubation with target protein (bait protein): (i) The target protein is either a recombinant protein with tags, such as GST, biotin, flag, His, located at its N or C terminal, or it is labeled with a fluorescent dye. (ii) It could be either a target protein for purification or a total lysate containing the target protein.
7) Washing: Remove any unbound target protein and reduce background noise.
8) Detection and visualization: Usually use tag secondary antibodies, then carry out enzyme-linked colorimetric reactions, chemiluminescence, or fluorescent detection methods to visualize the interaction.
Tabe 1. Various types of target proteins require distinct detection strategies
| Types of target proteins | Strategies |
|---|---|
| Unlabeled target protein | Enzyme-labeled target specific antibody |
| Radiolabeled target protein | Directly exposed the film |
| Biotinylated target protein | Enzyme-labeled streptavidin antibody |
| Tagged-fusion target protein | Tag-specific antibody |
9) Analysis and interpretation: The developed signals are analyzed and compared by imaging, densitometry, or other quantification methods.
10) Report delivery: The report will be delivered with a high level of quality, accuracy and within a fast turnaround.
Figure 1. A flowchart for Far-Western blot [1,2].
The complete renaturation of proteins on the membrane is also crucial for subsequent PPI assays. Rest assured, Creative Proteomics has developed methods to effectively restore protein folding. Importantly, the team at Creative Proteomics possesses advanced expertise in purifying target proteins using sophisticated techniques such as affinity chromatography to meet your specific project needs.
Experimental Control Design
In order to improve the accuracy of the results, appropriate experimental controls need to be set up. For example, if the bait protein is a GST-fusion protein, set up a separate GST tag experimental group as a negative control to exclude the possibility of non-specific binding of the GST tag itself to the target protein on the membrane.
Advantages of Far-WB
1) The experimental repeatability is commendable.
2) High-throughput, analyze multiple samples at once.
3) The molecular weight of interacting proteins can be determined instantly.
4) Cost-effective.
Disadvantages of Far-WB
- The experimental process involves multiple washing steps, making weak interactions difficult to detect.
- The detection of interactions becomes challenging when the prey protein is present at low concentrations in the tissue.
- Experiments may involve protein denaturation and renaturation and cannot detect interactions that depend on native structure.
Creative Proteomics also provide other methods for PPI study
1) Co-IP.
2) Crosslinking Protein Interaction Analysis.
3) Pull-Down Assay.
4) Label Transfer Protein Interaction Analysis.
5) BioID-MS Service.
6) Chemical Cross-linking Mass Spectrometry (CX-MS) Service.
7) Tandem Affinity Purification (TAP)-MS Service.
8) TurbolD Service.
How to place an order
The provision of comprehensive support tailored to your specific requirements for Far-Western Blot Analysis Service is our area of expertise. Please feel free to contact us via email whenever you need to discuss your specific requirements. Our customer service representatives are available 24 hours a day, from Monday to Sunday.
References
- Wu Y, Li Q, Chen XZ. Detecting protein-protein interactions by Far western blotting. Nature Protocols. 2007;2(12):3278-84.
- Krauspe V, Fahrner M, Spät P, et al. Discovery of a small protein factor involved in the coordinated degradation of phycobilisomes in cyanobacteria. Proc Natl Acad Sci U S A. 2021 Feb 2;118(5): e2012277118.
Detection of Protein–Protein Interactions by Far-Western Blotting
Journal: Protein Blotting and Detection
Published: 2009
Abstract
Far-western blotting is a convenient method to characterize protein–protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a nonantibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or the absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein–protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylationdependent manner.
Figure 1. Comparison of western and far-western blotting.
(A) Schematic representation of both methods. Box, bait protein and protein X interact via modular interaction domain and interaction motif. Left: western blotting uses antibodies raised against a target protein X to detect its presence. Middle and right: far-western blotting uses a protein probe containing a modular interaction domain or a short interaction motif, fused to a tag moiety (e.g., GST), to detect protein X based on presence of its binding sites. Specific interaction of far-western blotting is visualized by antibody-based detection (middle) or by direct labeling using isotope or an enzyme-conjugated affinity reagent, e.g., glutathione–HRP (right).
(B) Application of both methods. Breast cancer patient-derived samples (tumor 1–12) were immobilized on a membrane, and then identical membranes were probed with anti-EGFR antibody (left, western blotting) or GST-PI3K SH2 domain fusion (right, far-western blotting). Whereas both results look similar, interpretation of the results is somewhat different; the western result indicates a presence of EGFR in sample lane 5, 6, and 12, while the far-western result indicates a presence of PI3K SH2 domain recognition sites on a protein which is likely to be EGFR (arrow). In addition, the far-western probe also detected an uncharacterized PI3K SH2-binding protein (asterisk) that can potentially distinguish tumor 6 from 5 and 12, not otherwise detected by western blotting.
