Abstract
The introduction of fluorescent detection systems has revolutionized the applicability of Western blotting for quantitative protein expression analyses. The fundamental premise behind fluorescent Western blotting is the combination of distinct fluorescent dye-conjugated secondary antibodies and high performance digital imaging solutions in which the fluorescence signal is directly proportional to the amount of protein enabling quantitative measurements and simultaneous detection of several target proteins. This aspect of Western blotting is now widely used, especially in preclinical research, to detect quantitative changes in protein levels and phosphorylation status between experimental groups. This chapter provides a detailed step-by-step guide for best practice procedures during the entire process from sample preparation, SDS polyacrylamide gel electrophoresis to electrotransfer of proteins and highlights approaches that can be applied to increase data output.
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References
Renart J, Reiser J, Stark GR (1979) Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci U S A 76(7):3116–3120. https://doi.org/10.1073/pnas.76.7.3116
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76(9):4350–4354. https://doi.org/10.1073/pnas.76.9.4350
Elfving B, Müller HK, Oliveras I et al (2019) Differential expression of synaptic markers regulated during neurodevelopment in a rat model of schizophrenia-like behavior. Prog Neuro-Psychopharmacol Biol Psychiatry 95:109669. https://doi.org/10.1016/j.pnpbp.2019.109669
Müller HK, Wegener G, Popoli M et al (2011) Differential expression of synaptic proteins after chronic restraint stress in rat prefrontal cortex and hippocampus. Brain Res 1385:26–37. https://doi.org/10.1016/j.brainres.2011.02.048
Messa M (2018) Preparation of Synaptosomes from mammalian brain by subcellular fractionation and gradient centrifugation. Methods Mol Biol 1847:13–22. https://doi.org/10.1007/978-1-4939-8719-1_2
Müller HK, Wegener G, Liebenberg N et al (2013) Ketamine regulates the presynaptic release machinery in the hippocampus. J Psychiatr Res 47(7):892–899. https://doi.org/10.1016/j.jpsychires.2013.03.008
Ahmed S, Holt M, Riedel D et al (2013) Small-scale isolation of synaptic vesicles from mammalian brain. Nat Protoc 8(5):998–1009. https://doi.org/10.1038/nprot.2013.053
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Müller, H.K. (2022). A Guide to Analysis of Relative Synaptic Protein Abundance by Quantitative Fluorescent Western Blotting. In: Dahlmanns, J., Dahlmanns, M. (eds) Synaptic Vesicles. Methods in Molecular Biology, vol 2417. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1916-2_7
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DOI: https://doi.org/10.1007/978-1-0716-1916-2_7
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