25 Proteomics, Genomics & Microarrays


As with HKP normalisation, experiments to validate the positive and negative controls, and expression level test and linear range determination control experiments are required. However, once a sample dilution series has been established, running a single blot and gel can serve as the basis for all control experiments and is not impacted by the behaviour of a particular protein, unlike normalisation with HKPs which requires a new set of control experiments to be performed to optimise the signal or when experimental conditions change.


Stain-Free Total Protein Normalisation


is a simple and effi cient approach TPN can also be conducted using stain-free methods, which were introduced to overcome issues related to different membranes having different sensitivities to the stains used in TPN and provides a method to visualise the gel and blot with an imaging process that does not interfere with downstream immunodetection steps [8]. Stain-free technology also avoids the need for additional staining steps that would otherwise be required to visualise strong total protein signals with low background (Figure 2).


Transparency and meeting new


publication guidelines In line with recent discussions regarding the accuracy of normalisation in western blotting, several scientifi c journals, including the Journal of Biological Chemistry, have since updated their submission guidelines [10]. These show an increasing preference for TPN over HKP normalisation methods and reinforce the importance of transparency and reproducibility when reporting results from experimental data. Consequently, it is even more important for researchers to carefully consider their choice of normalisation method. Adhering to updated journal guidelines whilst maintaining the highest standards of experimental best practice and transparency when reporting data will increase researchers’ confi dence in their results, providing clearer insights into their investigations.


References


1. Oh K & Turner L (2018) The how and why of normalizing your western blots, https://www.bioradiations. com/the-how-and-why-of-normalizing-your-western-blots/


2. Bio-Rad, Total Protein Normalization. https://www.bio-rad.com/en-uk/applications-technologies/total- protein-normalization?ID=PODYJQRT8IG9


3. Fosang A J & Colbran R J (2015) Transparency Is the Key to Quality. The Journal of biological chemistry, 290(50), 29692–29694. https://doi.org/10.1074/jbc.E115.000002 4. Oh, Western Blot Normalization Methods. https://info.bio-rad.com/western-blotting-webinar-LP1.html


5. Janes K A (2015) An analysis of critical factors for quantitative immunoblotting. Science Signaling, 8(371), rs2. https://doi.org/10.1126/scisignal.2005966


6. Nie X et al (2017) An appropriate loading control for western blot analysis in animal models of myocardial ischemic infarction, Biochemistry and biophysics reports, 12, 108-113. https://doi.org/10.1016/j. bbrep.2017.09.001


7. Hu X et al (2016) Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better “housekeeper”. Oncotarget, 7(41), 66679–66688. https://doi.org/10.18632/oncotarget.11439


Figure 2: Comparison between high background and low background signals in gels with (left) and without (right) Stain-Free technology. Taken from Oh and Turner [1].


Visualisation of total protein on a gel or membrane in stain-free TPN is enabled through a proprietary trihalo compound that is directly embedded into the polyacrylamide gel. Following UV activation, the compound enhances natural protein fl uorescence by covalently binding to tryptophan residues, enabling visualisation of total protein without the need for staining and destaining steps (Figure 3).


8. Wang (2021) Stain-Free Western Blotting: Faster Results, Better Data, https://www.bioradiations.com/ stain-free-western-blotting-faster-results-better-data/


9. Hammond et al (2020) A Method for Greater Reliability in Western Blot Loading Controls: Stain-Free Total Protein Quantitation, https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6360.pdf


10. Collecting and Presenting Data, Journal of Biology Chemistry. https://jbcresources.asbmb.org/ collecting-and-presenting-data


About the authors


Katie Schaefer earned a PhD in Biochemistry studying gene regulation by transcription factor p53 in response to DNA oxidative stress. She began her career as a fi eld applications scientist with Bio-Rad Laboratories before moving into Global Marketing for chromatography and digital imaging products.


Figure 3: Exposure to UV light activates the embedded Stain-Free compound within Stain-Free gels, enabling visualisation of total protein. Taken from Wang [8].


In comparison to HKP normalisation, the overall workfl ow for stain-free TPN is considerably simplifi ed as running a single gel and blot serves as the basis for all control experiments. It also does not require any additional buffers or reagents, making it compatible with existing western blot workfl ows. Furthermore, stain-free TPN eliminates the issues related to the use of HKPs as loading controls as it is far more resistant to signal saturation within the common loading range for cell lysates. As a result, stain- free TPN establishes a linear and scalar response, allowing researchers to accurately quantify both the target protein and loading control within a linear dynamic range by normalising bands to total protein in each lane (Figure 4). In contrast, a scalar response is not observed with HKP expression, due in large part by combining chemiluminescence – a very sensitive signalling technique - with the immunodetection of a high abundance target making it challenging to differentiate any differences in lysate abundance. Correcting this issue often involves additional control experiments to optimise HKP antibody dilution ratios or using fl uorescent HKP antibodies with a wider linear dynamic range and signal response, which increases experimental effort and duration.


Kenneth Oh earned his PhD in bioorganic chemistry at UC Santa Barbara, where he synthesised fl uorescence-based peptide beacons that measured nucleic acid and protein binding events. On graduating, he cofounded a biotech company that reengineered virus capsids to serve as a drug delivery platform. He is currently the Marketing Manager for the Protein Quantitation Business Group at Bio-Rad Laboratories, responsible for the Electrophoresis and Western Blotting imagers, consumables, and reagents portfolio.


Deanna Woo is currently a Global Product Manager for Bio-Rad Laboratories’ Protein Quantitation Global Business division. She received her Bachelor of Science at San Francisco State University in Cellular and Molecular Biology, and held research roles at UCSF, Genentech, and Stanford. Her previous research work included studying the molecular mechanisms that regulate the development of primordial germ cells and research exploring hyperphosphorylation of tau in neurodegenerative diseases.


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The Spotlight could be on you!


Figure 4: Linearity comparison of Stain-Free total protein measurement and immunodetection of three housekeeping proteins in 10-50 μg of HeLa cell lysate. Adapted from Hammond et al [9].


Contact Gwyneth Astles on +44 (0)1727 855574 or email: gwyneth@intlabmate.com


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