Abstract
Cell signaling governs the basic functions of cells by molecular interactions that involve of many proteins. The abundance of signaling proteins can directly influence cellular responses to external signal, contributing to cellular heterogeneity. Absolute quantification of proteins is important for modeling and understanding the complex signaling network. Here, we introduce how to measure the amount of TGF-β signaling proteins using quantitative immunoblotting. In addition, we discuss how to convert the measurements of protein abundance to the quantities of absolute molecules per cell. This method is generally applicable to the absolute quantification of other proteins.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ho B, Baryshnikova A, Brown GW (2018) Unification of protein abundance datasets yields a quantitative Saccharomyces cerevisiae proteome. Cell Syst 6:192–205.e3
Gnann C, Cesnik AJ, Lundberg E (2021) Illuminating non-genetic cellular heterogeneity with imaging-based spatial proteomics. Trends Cancer 7:278–282
Wu L, Candille SI, Choi Y, **e D, Jiang L, Li-Pook-Than J, Tang H, Snyder M (2013) Variation and genetic control of protein abundance in humans. Nature 499:79–82
Aldridge BB, Burke JM, Lauffenburger DA, Sorger PK (2006) Physicochemical modelling of cell signalling pathways. Nat Cell Biol 8:1195–1203
Beck M, Schmidt A, Malmstroem J, Claassen M, Ori A, Szymborska A, Herzog F, Rinner O, Ellenberg J, Aebersold R (2011) The quantitative proteome of a human cell line. Mol Syst Biol 7:549
Gillet LC, Leitner A, Aebersold R (2016) Mass spectrometry applied to bottom-up proteomics: entering the high-throughput era for hypothesis testing. Annu Rev Anal Chem (Palo Alto, Calif) 9:449–472
Ankney JA, Muneer A, Chen X (2018) Relative and absolute quantitation in mass spectrometry-based proteomics. Annu Rev Anal Chem (Palo Alto, Calif) 11:49–77
Torres NP, Ho B, Brown GW (2016) High-throughput fluorescence microscopic analysis of protein abundance and localization in budding yeast. Crit Rev Biochem Mol Biol 51:110–119
Chong YT, Koh JL, Friesen H, Duffy SK, Cox MJ, Moses A, Moffat J, Boone C, Andrews BJ (2015) Yeast proteome dynamics from single cell imaging and automated analysis. Cell 161:1413–1424
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 76:3116–3120
Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O'Shea EK, Weissman JS (2003) Global analysis of protein expression in yeast. Nature 425:737–741
Colak S, Ten Dijke P (2017) Targeting TGF-beta Signaling in cancer. Trends Cancer 3:56–71
van der Kraan PM (2017) The changing role of TGFbeta in healthy, ageing and osteoarthritic joints. Nat Rev Rheumatol 13:155–163
Massague J (1998) TGF-beta signal transduction. Annu Rev Biochem 67:753–791
Shi Y, Massague J (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113:685–700
Hill CS (2016) Transcriptional control by the SMADs. Cold Spring Harb Perspect Biol 8:a022079
McDonough AA, Veiras LC, Minas JN, Ralph DL (2015) Considerations when quantitating protein abundance by immunoblot. Am J Physiol Cell Physiol 308:C426–C433
Janes KA (2015) An analysis of critical factors for quantitative immunoblotting. Sci Signal 8:rs2
Clarke DC, Liu X (2010) Measuring the absolute abundance of the Smad transcription factors using quantitative immunoblotting. Methods Mol Biol 647:357–376
Pillai-Kastoori L, Schutz-Geschwender AR, Harford JA (2020) A systematic approach to quantitative Western blot analysis. Anal Biochem 593:113608
Acknowledgments
This work was supported by a grant to Z.Z. from the Federal Ministry of Education and Research (BMBF, Germany) within the e:Bio SyBioT project (031A309).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Deng, D., Zi, Z. (2022). Absolute Quantification of TGF-β Signaling Proteins Using Quantitative Western Blot. In: Zi, Z., Liu, X. (eds) TGF-Beta Signaling. Methods in Molecular Biology, vol 2488. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2277-3_1
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2277-3_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2276-6
Online ISBN: 978-1-0716-2277-3
eBook Packages: Springer Protocols