Abstract
The choice of an antibody for a protein-based research study is one of the most crucial steps in any project. Seemingly straightforward, the process is actually quite nuanced and filled with potential pitfalls. In this chapter, we will discuss five major topics that require consideration in the antibody selection process. These include overall study objectives and resources, details of both species and clonality, suitability in applications, and available detection methods. Each section will provide background information on the topic as well as specifics of antibody use in the laboratory. This chapter may be used as a guide to help vet antibody candidates for your project so you can stain with confidence.
Key words
- Antibody
- Validation
- Specificity
- Species
- Clonality
- Western blot
- Immunostaining
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References
Coultrap SJ, Nixon KM, Alvestad RM, Valenzuela CF, Browning MD (2005) Differential expression of NMDA receptor subunits and splice variants among the CA1, CA3 and dentate gyrus of the adult rat. Mol Brain Res 135:104–111
Baker M (2015) Reproducibility crisis: blame it on the antibodies. Nature (London) 521:274–276
Marx V (2013) Finding the right antibody for the job. Nat Methods 10:703–707
Yu W, Hill WG (2013) Lack of specificity shown by P2Y6 receptor antibodies. Naunyn Schmiedeberg’s Arch Pharmacol 386:885–891
Herrera M, Sparks MA, Alfonso-Pecchio AR, Harrison-Bernard LM, Coffman TM (2013) Lack of specificity of commercial antibodies leads to misidentification of angiotensin type 1 receptor protein. Hypertension 61:253–258
Milner R, Wombwell H, Eckersley S et al (2013) Validation of the BRCA1 antibody MS110 and the utility of BRCA1 as a patient selection biomarker in immunohistochemical analysis of breast and ovarian tumours. Virchows Arch 462:269–279
Baek JH, Darlington CL, Smith PF, Ashton JC (2013) Antibody testing for brain immunohistochemistry: brain immunolabeling for the cannabinoid CB(2) receptor. J Neurosci Methods 216:87–95
Goodpaster T, Randolph-Habecker J (2014) A flexible mouse-on-mouse immunohistochemical staining technique adaptable to biotin-free reagents, immunofluorescence, and multiple antibody staining. J Histochem Cytochem 62(3):197–204
Goelz SE, Nestler EJ, Chehrazi B, Greengard P (1981 Apr) Distribution of protein I in mammalian brain as determined by a detergent-based radioimmunoassay. Proc Natl Acad Sci U S A 78(4):2130–2134
Helsby MA, Fenn JR, Chalmers AD (2013) Reporting research antibody use: how to increase experimental reproducibility. F1000Res 2:153
Algenas C, Agaton C, Fagerberg L et al (2014) Antibody performance in western blot applications is context-dependent. Biotechnol J 9:435–445
Burry RW (2011) Controls for immunocytochemistry: an update. J Histochem Cytochem 59:6–12
Egelhofer TA, Minoda A, Klugman S et al (2011) An assessment of histone-modification antibody quality. Nat Struct Mol Biol 18:91–93
Murphy RM, Lamb GD (2013) Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes. J Physiol 591:5823–5831
Kurien BT, Dorri Y, Dillon S, Dsouza A, Scofield RH (2011) An overview of Western blotting for determining antibody specificities for immunohistochemistry. Methods Mol Biol 717:55–67
Gilda JE, Ghosh R, Cheah JX, West TM, Bodine SC, Gomes AV (2015) Western blotting inaccuracies with unverified antibodies: need for a Western Blotting Minimal Reporting Standard (WBMRS). PLoS One 10:e0135392
Schuster C, Malinowsky K, Liebmann S et al (2012) Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues. Histopathology 60:E37–E50
Sawicka M, Pawlikowski J, Wilson S et al (2013) The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding. PLoS One 8:e53313
Saper CB (2009) A guide to the perplexed on the specificity of antibodies. J Histochem Cytochem 57:1–5
Gore AC (2013) Editorial: antibody validation requirements for articles published in endocrinology. Endocrinology 154:579–580
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Archuleta, A., Browning, M.D. (2023). Top Five Considerations When Choosing an Antibody. In: Kalyuzhny, A.E. (eds) Signal Transduction Immunohistochemistry. Methods in Molecular Biology, vol 2593. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2811-9_1
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DOI: https://doi.org/10.1007/978-1-0716-2811-9_1
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