The Advancing Applications of Western Blotting

Since they were first developed in the late 1970s, western blots (WBs) have come to play a key role in many clinical and research laboratories and are now the most widely used protein detection technique.1 However, despite their widespread use, WBs are time consuming, requiring precision and well-trained operators, and are beset with limitations. Applications can also be limited by the availability and quality of primary antibodies to the protein of interest. As such, research continues into the development of novel techniques and technologies that can improve the accuracy and efficiency of this core technique. The Advancing Applications ofWestern Blotting Sponsored by Applications of WBs WBs have applications in both clinical and research settings,1 and can be used for a range o f applications including:2 Protein–protein interaction detection: Far-western blotting validates binding partners by probing with a labeled protein. Post-translational modification analysis: WBs can detect modifications including phosphorylation, ubiquitination and glycosylation using modification-specific antibodies. Epitope mapping: Using fragments or overlapping peptides of the antigen blotted onto a membrane, WBs can reveal antibody binding sites. Disease diagnosis: WBs can be used to detect disease-associated proteins, including viral and autoimmune markers for diseases such as tuberculosis, lupus and HIV. Antibody characterization: WBs can confirm antibody specificity, cross-reactivity and target recognition. A new lease of life Despite the challenges of a WB, it remains a lab staple, and novel developments to improve the technique continue to emerge. References 1. Moritz CP. 40 years Western blotting: A scientific birthday toast. J Proteomics. 2019;212:103575. doi: 10.1016/j.jprot.2019.103575 2. Meftahi GH, Bahari Z, Zarei Mahmoudabadi A, Iman M, Jangravi Z. Applications of western blot technique: From bench to bedside. Biochemistry and Molecular Biology Education. 2021;49(4):509-517. doi:10.1002/bmb.21516 3. Hughes AJ, Spelke DP, Xu Z, Kang CC, Schaffer DV, Herr AE. Single-cell western blotting. Nat Methods. 2014;11(7):749-755. doi: 10.1038/nmeth.2992 4. Fosang AJ, Colbran RJ. Transparency is the key to quality. J Bio Chem. 2015;290(50):29692-29694. doi: 10.1074/jbc.e115.000002 The challenges and limitations of traditional WBs Not as sensitive as other techniques due to relatively high (> 10 μg) lower limit of detection Require functional, high-quality specific primary antibodies Depend on operator capability and skill for accurate sample preparation Time consuming multistep protocol can be prone to errors Low throughput – only able to detect one protein at a time Difficult to standardize and accurately quantify – not easily reproducible Single-cell WB Single cell WBs can detect variations in protein expression between individual cells, using a thin layer of polyacrylamide gel and a series of microwells, each of which contain a single cell. Cells are lysed in situ, then gel electrophoresis, blotting and antibody probing are performed as in a traditional WB.3 Allows multiplexing of different targets in the same cell Can be used for extremely precious or limited clinical samples Can increase accuracy and differentiate between protein isoforms by analyzing molecular mass and antibody binding3 Can analyze thousands of cells individually in complex populations Automated WB processors One of the main drawbacks of WBs is the long hands-on time: multiple washes and incubations, sometimes spanning days. Automated WB processors are now commercially available, drastically reducing the time-cost of WBs. Reduce variability of hands-on protocols Faster assay time – hours rather than days Microchip electrophoresis Microchip electrophoresis uses capillary electrophoresis rather than standard SDS-PAGE to separate cellular proteins. Multiple replicates of the same protein sample are loaded into individual tracks on a microchip, then captured on a polyvinylidene difluoride (PVDF) membrane for subsequent immunoassay stages. Higher sensitivity with better resolution Allows for measurement of multiple targets from a single cell No need for blocking step, resulting in shorter analysis time Easy to multiplex, allowing multiple proteins to be analyzed simultaneously Total protein normalization A traditional WB uses housekeeping proteins (HKP) such as β-actin to standardize blots and help analyze the level of target protein expression. However, HKP are assumed to be constant, but they can be affected by experimental variations and expressed at much higher levels that target proteins, resulting in oversaturate control bands. Measures total cellular protein levels, accounting for the intensity of all proteins Shows improved linearity between target and control proteins Is more robust and less susceptible to variation during processing than HKP Meets publishing guidelines for quantitative WBs, ensuring high-quality, reliable results4 As WB technology advances, the challenges in efficiency, reproducibility and sensitivity that have plagued WB are being overcome. In addition, improved antibody production, enhanced chemiluminescent and fluorescent imagers add further advances, ensuring that WB is likely to continue to be a cornerstone of many labs for the foreseeable future. Written by Kate Harrison, PhD. Designed by: Erin Lemieux