Journal article
Quantifying cytoskeletal protein interactions with far Western blotting
Frontiers in molecular biosciences, Vol.13, p.1761723
02/12/2026
DOI: 10.3389/fmolb.2026.1761723
PMCID: PMC12935617
PMID: 41767045
Abstract
The study of cytoskeletal proteins has been limited by some of their intrinsic properties, such as fixed intracellular localization and insolubility, which limit the implementation of many protein–protein interaction assays. Far Western blotting is a powerful biochemical technique used to detect and quantify protein–protein interactions on a membrane, addressing limitations in cytoskeletal research. This method combines traditional Western blotting with protein overlay assays to identify and characterize binding partners in complex samples. Here, we present a detailed protocol for the use of far Western blotting to probe keratin–keratin interactions, including instructions for protein sample preparation, electrophoretic separation, membrane transfer, protein labeling, buffer dilutions, and detection steps. We further extend the use of far Western blotting to compare relative binding and enhance assay sensitivity by incorporating a Western blot as a loading control. This approach enables the analysis of samples that are too dilute for reliable membrane staining. Key considerations for enhancing specificity and sensitivity are discussed, along with troubleshooting tips. This approach offers a versatile tool for studying protein interaction networks, providing valuable insights into cellular signaling and molecular mechanisms, and can be conducted in any laboratory capable of performing a Western blot.
Details
- Title: Subtitle
- Quantifying cytoskeletal protein interactions with far Western blotting
- Creators
- Marcelo M. Melo (Author) - University of IowaElizabeth S. Phillippi (Author) - University of IowaMark Schultz (Author) - University of IowaSusan Q Shen - University of IowaHatem El-Shanti (Corresponding Author) - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Frontiers in molecular biosciences, Vol.13, p.1761723
- DOI
- 10.3389/fmolb.2026.1761723
- PMID
- 41767045
- PMCID
- PMC12935617
- NLM abbreviation
- Front Mol Biosci
- ISSN
- 2296-889X
- eISSN
- 2296-889X
- Publisher
- Frontiers Media SA
- Copyright
- © 2026 Melo, Phillippi, Schultz, Shen and El-Shanti.
- Grant note
- University of Iowa Stead Family Children's Hospital
The author(s) declared that financial support was received for this work and/or its publication. This research received no external funding; it was supported by multiple internal grants and start-up and discretionary funds from the University of Iowa Stead Family Children's Hospital.
- Language
- English
- Date published
- 02/12/2026
- Academic Unit
- Stead Family Department of Pediatrics; Medical Genetics and Genomics
- Record Identifier
- 9985224971702771
Metrics
6 Record Views