This often requires regeneration of the membrane by stripping off the antibodies and reprobing 19. However, chemiluminescence is not amenable to multi-target detection when using antibodies with low specificity or for proteins that are very close in molecular weight with overlapping signals. Multiplexed target detection from chemiluminescent western blots is feasible when working with highly specific antibodies against targets that differ in molecular weight by at least five to ten kilodaltons (kDa). With limiting sample availability to assess components of microdissected tissue, sorted cell subpopulations, exosomes and primary cells cultured from small tissue specimens, the requirement to maximize quantity while preserving the quality of data is paramount 17, 18. However, a complete and concise guide to the workflow highlighting the importance of reagent selection, data workup and analysis remains elusive. These highly viewed and well cited articles support the need for updated guidance in producing quantitative western blot data.
#Western blot results of fish full#
Recent articles have dissected and contrasted the old practices passed down from the early days of western blotting to offer revised methodologies that take full advantage of the currently available state-of-the-art instruments and reagents to achieve accurate, precise and reproducible data 14, 15, 16. The associated retractions from high profile articles 10, 11, 12, 13 support the unfounded reputation of western blotting as a difficult, frustrating and non-reproducible technique that is semi-quantitative at best. However, the original qualitative techniques and assumptions that have been passed down through generations of lab members were not formally updated to permit the production of semi-quantitative western blot data thus giving poor reproducibility and misleading interpretations 7, 8, 9. This permitted the generation of semi-quantitative data from chemiluminescence.
During this time, the first camera-based imaging systems were introduced which enabled automated detection over a much broader linear dynamic range of about three to four orders of magnitude 6. Since the linear quantitative range of film is only about an order of magnitude and very challenging to assess saturation by eye, the results even between technical replicates were difficult to reproduce 5. Over the following two decades, as reagent and antibody technologies evolved, scientists attempted to produce quantitative results initially by scintillation counting and film densitometry 3, 4. Western blotting was first introduced in 1979 as a means of detecting a target protein in a sample with antibodies and detection using film 1, 2.
#Western blot results of fish how to#
The data reveal when and how to apply these detection methods to achieve reproducible data with a stepwise approach to data processing for quantitative analysis. A methodology to optimize and produce excellent, quantitative western blot results with rigorous data analysis from membranes probed with both fluorescent and chemiluminescent antibodies is described. Fluorescence reagent and detection solutions are the latest innovation in western blotting but there remains questions and debate concerning their relative sensitivity and dynamic range versus chemiluminescence. The development of sensitive, camera-based detection technologies coupled with an updated technical approach permits the production of reproducible, quantitative data. Chemiluminescence coupled with film-based detection was traditionally the gold standard for western blotting but accurate and reproducible quantification has been a major challenge from this methodology. Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance.
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