CiteAb’s introductory guide: western blotting alternatives
10
Min Read
In this blog:
- We outline some common alternatives to western blotting
- How is traditional western blotting being automated?
- The importance of antibody validation
Western blotting has been a mainstay in molecular biology for many years due to its power to separate and identify proteins. In fact, CiteAb data shows it is the most cited immuno-assay. So, why seek alternatives?
Although the western blotting process can be set up in almost any lab, it can be time consuming and it may be tricky to get reliable results consistently due to the many steps involved [1].
In this blog, we will look at the automation and improvement of traditional western blotting, as well as emerging alternatives to the technique, namely: capillary gel electrophoresis and capillary isofocusing.
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Capillary gel electrophoresis
Unlike the traditional protein separation electrophoresis techniques using an SDS-PAGE gel (size based separation), this technique is performed in a capillary tube with a polymer liquid or gel. Similarly to gel electrophoresis, ions are separated by electrophoretic mobility after application of a voltage (aka. still size separation).
The design of capillaries ensures high voltages can be applied with minimal heat generation, enabling faster analysis times as well as high separation efficiency [2]. Many different sieving matrices can be used in this method, each with different benefits [3].
Significantly, this technique combines blotting and separation – this means protein is captured as it migrates from the capillary, minimising the difficulties of multistep western blotting.
An example:
An example of this technique used in action is the SimpleWestern technology from ProteinSimple. We have observed an overall growth in citations in CiteAb data for this technology, with a peak in 2020.
With this technology, after separation proteins are attached to the capillary wall using chemical crosslinking, activated by ultraviolet. Blotting steps are then done automatically, and the specific antibodies for detection are applied conjugated to horseradish peroxidase. This tag enables an electropherogram trace or virtual gel image to be produced [4].
Significantly, all steps are combined into one automated process that is fast, conserves sample, and allows for analysis using multiple different antibodies (up to 90!)
Possible downsides of this technique may include finding compatible antibodies compared to traditional techniques, and costs – although the time saved may outweigh initial investment.
Alternatively, academics can carry out their own capillary electrophoresis in the lab [5].
Capillary isofocusing
With capillary isofocusing, proteins are separated by isoelectric point rather than by size. In short, due to a pH gradient being established across the gel, the protein will stop migrating when it reaches the pH corresponding to its pI.
The benefits of this method include a higher resolution, faster sample analysis and sample conservation [6].
An example:
SimpleWestern charge based separation is an example of a technique using capillary isofocusing. CiteAb data shows fewer citations for this method than size based equivalents.
This method has high sensitivity and you can observe very small differences in proteins being analysed, such as the differences brought about by post translational modifications [7].
This technique can also be carried out in individual labs [8].
Further new methods, not mentioned in our overview, include: microfluidic systems, single cell resolution western blot, far western blotting, diffusion blotting and microchip electrophoresis. Although these methods are less frequently used they are still worth investigating – do look out for a blog in the future covering these.
Automation of traditional western blotting
More traditional western blotting is not out of the game, despite alternative methods being developed. It is still a widely used technique, and thanks to technological advancements and innovations, it is now more efficient[9].
Improvements besides automation include the methods by which proteins are transferred from the SDS-PAGE gel to the membrane (e.g. vacuum blotting or centrifuge blotting), pre-made gels with better separation abilities and shelf lives, and improved sensitivity through new chemiluminescent reagents and imagers being developed, for example.
Most significantly, by automating western blotting some of the main drawbacks of traditional western blotting are overcome. Importantly, reproducibility is improved as the manual multi-step process is avoided, thereby saving time for researchers.
Below we list a few examples of automated western blotting machines from a range of suppliers which help make the traditional western blotting process more efficient.
Examples:
Invitrogen: The automated western machine by invitrogen automates steps of blocking, washing and antibody incubation.
Precision biosystems: The BlotCycler™ automates traditional western blotting steps, you can also reuse your membrane and no special reagents are required.
Genscript: With eZwest™ automated blocking, washing and incubation steps are carried out.
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It all comes back to antibodies
No matter the improvements made to western blotting techniques, if the antibodies used are not of high enough quality, then reliable results will not be produced. It is important that antibodies are validated for specificity, and for the application of western blotting or alternative applications.
Without proper validation, you can get non-specific protein binding, ultimately producing incorrect results. You can read our guide on how to find an antibody, covering these important considerations and how to approach the purchasing process.
In CiteAb, you can filter by application when searching for antibodies, helping you narrow down antibodies to choose for western blotting, and we recently added filters for Simple Western Size and isoelectric focusing (“SW-Size” and “SW-IEF”). We also include validation information and display published images, so you can see if the antibody has produced a nice western blot before purchasing.
Image of a western blot using a Miltenyi Biotec CD133/1 antibody. Collected and cropped by CiteAb under a CC-BY license: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747371/
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Find antibodies for western blotting using the CiteAb search:
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References:
[1] Mishra, M., Tiwari, S. and Gomes, A.V. (2017). Protein purification and analysis: next generation Western blotting techniques. Expert Review of Proteomics, [online] 14(11), pp.1037–1053. doi:10.1080/14789450.2017.1388167.
[2]Righetti, P.G., Sebastiano, R. and Citterio, A. (2012). Capillary electrophoresis and isoelectric focusing in peptide and protein analysis. PROTEOMICS, 13(2), pp.325–340. doi:10.1002/pmic.201200378.
[3] Zhu, Z., Lu, J.J. and Liu, S. (2012). Protein separation by capillary gel electrophoresis: A review. Analytica Chimica Acta, [online] 709, pp.21–31. doi:10.1016/j.aca.2011.10.022.
[4] www.proteinsimple.com. (n.d.). Simple Western Assays :: ProteinSimple. [online] Available at: https://www.proteinsimple.com/simple_western_assays.html [Accessed 5 May 2022].
[5] Anderson, G.J., M Cipolla, C. and Kennedy, R.T. (2011). Western blotting using capillary electrophoresis. Analytical Chemistry, [online] 83(4), pp.1350–1355. doi:10.1021/ac102671n.
[6] Righetti, P.G., Sebastiano, R. and Citterio, A. (2012). Capillary electrophoresis and isoelectric focusing in peptide and protein analysis. PROTEOMICS, 13(2), pp.325–340. doi:10.1002/pmic.201200378.
[7] www.proteinsimple.com. (n.d.). Simple Western Assays Utilizing Charge-Based Separation :: ProteinSimple. [online] Available at: https://www.proteinsimple.com/simple_western_charge_assays.html [Accessed 5 May 2022].
[8] Aspinall-O’Dea, M., Pierce, A., Pellicano, F., Williamson, A.J., Scott, M.T., Walker, M.J., Holyoake, T.L. and Whetton, A.D. (2015). Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines. Nature Protocols, [online] 10(1), pp.149–168. doi:10.1038/nprot.2015.007.
[9]Mishra, M., Tiwari, S. and Gomes, A.V. (2017). Protein purification and analysis: next generation Western blotting techniques. Expert Review of Proteomics, [online] 14(11), pp.1037–1053. doi:10.1080/14789450.2017.1388167.
[10] www.thermofisher.com. (n.d.). Bandmate Automated Western Blot Processor – US. [online] Available at: https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/bandmate-automated-processor.html [Accessed 5 May 2022].
