In its early days, the assay delivered information in a strict binary of ‘yes’ or ‘no’ – protein present or absent. In recent years, however, improved western blot technology and technique have allowed an exciting quantitative element to emerge. Researchers can now reliably measure relative changes in protein expression and abundance.īut with this shift towards quantitative understanding comes a movement by major journals, funding agencies, and professional scientific societies to uphold the highest standards of data integrity for publication. Their updated guidelines for immunoblotting images come alongside guardrails against data manipulation, intentional or inadvertent, in a growing age of generative AI and accessible image editing software. Quantitative Western Blotting Requires Normalization So, what does a “publication-ready” western blot mean today to the top journals in the field, and what tips, tools, and products exist to help researchers get there? This comprehensive guide will help steer you through the process. With a quantitative western blot, researchers can gain valuable insight into relative protein expression. Quantitative western blotting requires careful interpretation, particularly around non-uniformities. Variability in western blotting occurs most often with unequal protein concentrations, inconsistent sample loading onto the gel, and irregularities during transfer – all normal, human realities of the process. Normalization accounts for those realities by distinguishing experimental variability from true biological changes in protein expression. Skillful normalization ultimately encourages accuracy and reproducibility in the literature. Right now, methods for immunoblotting normalization and quantitation across the field vary more than many experts would like.Įditorial staff of the field-leading publication Journal of Biological Chemistry named acceptable presentation and quantitation of western blots as one of three “ major gaps in overall data reporting” among submissions. Their recently issued revised guidelines for blot reporting reflect a wider push towards total protein normalization (TPN) in lieu of another popular normalization strategy using housekeeping proteins (HKP). Let’s talk through some of the downsides of housekeeping protein protocols and advantages of total protein normalization. Housekeeping Proteins (HKP) Are Falling Out of Favor with Journalsįor decades, researchers have used housekeeping proteins like GAPDH, β-tubulin, β-actin, or cyclophilin B as internal loading controls for normalization. In this approach, changes in a sample’s target protein level can be expressed as a ratio to the chosen HKP. HKP is still the most-used method of western blotting normalization today. The problem with this protocol is that it assumes consistent HKP expression across experiments, and more and more studies have now confirmed that this just isn’t the case. HKP expression is more variable than constant, changing with cell type and developmental stage tissue age, type, and pathology post-transcriptional regulation experimental conditions and more (Table 1).
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