Far-Western Blot Service

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Far-Western Blot Service

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CD BioSciences provides the Far-Western Blot service, a molecular biology method based on Western Blot technology that can be used to detect protein-protein interactions in vitro, especially interaction assays that do not require the natural structure of the target protein.

We provide the Far-Western Blot technology that immobilizes target proteins on PVDF/NC membranes, uses bait proteins (known proteins) as probes to detect target proteins, and detects protein interactions based on the presence or absence of protein probe binding sites. This method can characterize protein-protein interactions involved in plant biological processes such as signal transduction, including interactions regulated by post-translational modifications.

Comparison of Western and Far-Western Blot. Figure 1. Comparison of Western and Far-Western Blot. (Jadwin, J. A., et al., 2015)

Service Content

Experimental content

  • Quantify proteins and separate them using SDS or native PAGE.
  • Transfer proteins from the gel to the membrane.
  • Denature and denaturation of proteins to ensure full exposure of protein binding sites on the membrane.
  • BSA buffer to seal the membrane.
  • Incubate membranes with purified bait proteins.
  • Detection of bait protein signals.
  • Image analysis and report delivery.

Probe protein detection service

Depending on the presence or absence of a marker or tag on the bait protein, various methods can be used to detect protein-protein interactions, including

  • Radioactive bait proteins are exposed to gel for direct detection of prey proteins.
  • Indirect detection uses antibodies specific to the bait protein, which is unlabeled.
  • Fusion-tagged bait proteins are indirectly detected using tag-specific antibodies and enzyme-labeled secondary antibodies.
  • Biotinylated bait proteins are indirectly detected with enzyme-coupled affinity proteins or streptavidin.

Optimization service of Far-Western Blot

  • Fusion proteins are synthesized under conditions without excessive degradation and insolubilization of the protein.
  • Monitoring the state of the fusion protein, both during and after purification.
  • Setting up specificity controls.

    • a) Probing with fusion protein mutants to disrupt interactions.

    b) Probing membranes with labeled GST.

  • Probing membranes with denaturation/reversion cycles to refold misfolded proteins into their natural conformation.

Sample Requirements

Sample type Details
Plant tissue 250-500mg/sample.
Cells 1×106 -1×107/sample.
Protein 100-150μg/sample, concentration ≥ 2 μg/μl.

Advantages of Far-Western Blot

  • Analyze protein binding domains.
  • Compare protein binding affinities.
  • Does not require specific antibodies.
  • Multiple tissue samples can be analyzed at once.
  • The molecular weight of interacting proteins can be determined immediately.

CD BioSciences offers Far-Western Blot services for studying plant protein-protein interactions. We can customize the experiments according to the needs of our clients and evaluate the potential factors that may affect the results. We are committed to providing reliable and reproducible data. Feel free to contact us anytime!

References

  1. Jadwin, J. A., et al. (2015). Detection and quantification of protein-protein interactions by far-western blotting. Methods in molecular biology (Clifton, N.J.). 1312, 379-398.
  2. Machida, K., & Mayer, B. J. (2009). Detection of protein-protein interactions by far-western blotting. Methods in molecular biology (Clifton, N.J.). 536, 313-329.

For research use only, not for clinical use.