Far-Western Blot Service

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Far-Western Blot Service

Far-Western Blot Service

Far-western blot is a molecular biological method for detecting protein-protein interactions. Far-Western blot is similar to Western blot. In Western blot experiments, specific antibodies (primary antibodies) are used to detect proteins on the membrane. The HRP-labeled secondary antibody is combined with the primary antibody, and the protein on the membrane is observed by development. In Far western blot, the target protein is fixed on the PVDF / NC membrane, and the bait protein (known protein) is used as a probe to detect the target protein on the membrane. The specific antibody incubation and detection are used to analyze the interaction between the target protein and the bait protein. Far-western blot technology can verify the interactions between known proteins or analyze the interactions between known and unknown proteins.

Creative Proteomics provides one-stop Far-Western Blot service to provide you with subcellular protein interaction analysis accelerating your project process.

The Process of Far-Western Blot Service

Gel ElectrophoresisProteins of different sizes are separated by SDS-PAGE or natural PAGE.
Transfer FilmAfter the proteins in the sample are separated on the gel, the proteins are transferred from the gel to the membrane. The purpose of this step is to attach the protein to the surface of the membrane, making the protein easier to detect. During the transfer process, we have strictly regulated operations to avoid contamination.
BlockingAfter the transfer, the entire membrane is generally closed with a heterologous protein to block non-specific binding sites. Commonly used blocking agents are skimmed milk powder, BSA and so on. It should be noted that the blocking agent may cross-react or disrupt the protein interaction to be studied in other ways. We will choose the right blocking agent based on our extensive experience.
Probe IncubationThe bait protein is incubated with the blocked membrane to bind the bait protein to the interacting protein on the NC membrane. After incubation, unbound bait proteins are washed away. In order to reduce the experimental background, we will choose purified protein as the probe.
Probe Protein DetectionThere are several strategies for detecting probe proteins:
  • Unlabeled bait protein → enzyme-labeled bait characteristic antibody → substrate reagent
  • Radiolabeled decoy protein → exposed to film
  • Biotinylated bait protein → enzyme-labeled streptavidin → substrate reagent
  • Fusion-labeled decoy protein → tag-specific antibody → enzyme-labeled secondary antibody → substrate reagent

Advantages of Far-Western Blot Protein Interaction Service:

  • This method has good repeatability.
  • Far-western blot can analyze multiple tissue samples at once.
  • Molecular weight of interacting proteins can be determined immediately.

Want to Know about Other Subcellular Protein-protein Interaction Analysis Techniques?

References

  1. Wu Y, Li Q, Chen X Z. Detecting protein–protein interactions by far western blotting. Nature protocols, 2007, 2(12): 3278.
  2. Edmondson D G, Roth S Y. Identification of protein interactions by far Western analysis. Current protocols in cell biology, 2000, 7(1): 17.2. 1-17.2. 10.

*For Research Use Only. Not used for clinical diagnosis and treatment.

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