Abstract
Western blotting is a highly adaptable biochemical assay that is used widely across cellular and molecular biology disciplines and has a diverse array of applications. This technique has been used for over four decades and remains a popular method for both protein detection and quantification. Identifying proteins of interest, such as metabotropic glutamatergic receptors (mGlu receptors), has proven beneficial for addressing key questions within the field. However, the promiscuity of mGlu receptor antibodies has yielded inconsistent results with high levels of nonspecific binding and background staining. In addition, mGlu receptors frequently form heterodimers or homodimers with other receptors. The overarching goal of this chapter is to discuss the theory of western blotting, applying of this technique for answering scientific questions regarding changes in mGlu receptor protein expression, and provide a standardized western blotting protocol for immunoblotting of these receptors. While other immunoblotting methods can also be used, this chapter contains general information that will aid researchers in the ability to conduct western blot experiments, especially those relating to mGlu receptor expression.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Perkel JM (2015) A history of the old West(ern blot). In: Science Magazine. Science/AAAS Custom Publishing Office, Athens
Gibbons J (2014) Western blot: protein transfer overview. N Am J Med Sci 6(3):158–159
Ninfa AJ, Ballou DP, Benor M (2009) Fundamental laboratory approaches for biochemistry and biotechnology, 2nd edn. Wiley, Hoboken, NJ, p 480
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
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
Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112(2):195–203
Gallo RC et al (1984) Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224(4648):500–503
Wilhelm BG et al (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344(6187):1023–1028
Anderson GJ, Cipolla CM, Kennedy RT (2011) Western blotting using capillary electrophoresis. Anal Chem 83(4):1350–1355
Damm K et al (2015) Improved northern blot detection of small RNAs using EDC crosslinking and DNA/LNA probes. Methods Mol Biol 1296:41–51
Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3(6):1077–1084
Brown T (1993) Southern blotting. Curr Protoc Mol Biol 21(1):2.9.1–2.9.20
Papoulas O (2001) Rapid separation of protein-bound DNA from free DNA using nitrocellulose filters. Curr Protoc Mol Biol Chapter 12:Unitas 12.8
Fields C et al (2019) Northern blot with IR fluorescent probes: strategies for probe preparation. Bio Protoc 9(8):e3219
Huang Q et al (2014) A non-radioactive method for small RNA detection by northern blotting. Rice (N Y) 7(1):26
Coorssen JR et al (2002) Quantitative femto- to attomole immunodetection of regulated secretory vesicle proteins critical to exocytosis. Anal Biochem 307(1):54–62
Murphy RM, Lamb GD (2013) Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes. J Physiol 591(23):5823–5831
Bass JJ et al (2017) An overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports 27(1):4–25
Conn PJ, Pin JP (1997) Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 37:205–237
Bortolotto ZA et al (1994) A molecular switch activated by metabotropic glutamate receptors regulates induction of long-term potentiation. Nature 368(6473):740–743
Balschun D et al (1999) A specific role for group I mGluRs in hippocampal LTP and hippocampus-dependent spatial learning. Learn Mem 6(2):138–152
Mameli M et al (2007) Rapid synthesis and synaptic insertion of GluR2 for mGluR-LTD in the ventral tegmental area. Science 317(5837):530–533
Niswender CM, Conn PJ (2010) Metabotropic glutamate receptors: physiology, pharmacology, and disease. Annu Rev Pharmacol Toxicol 50:295–322
Snyder EM et al (2001) Internalization of ionotropic glutamate receptors in response to mGluR activation. Nat Neurosci 4:1079–1085
Sanderson TM, Collingridge GL, Fitzjohn SM (2011) Differential trafficking of AMPA receptors following activation of NMDA receptors and mGluRs. Mol Brain 4:30
Lan JY et al (2001) Activation of metabotropic glutamate receptor 1 accelerates NMDA receptor trafficking. J Neurosci 21(16):6058–6068
Cheng J et al (2013) SNARE proteins are essential in the potentiation of NMDA receptors by group II metabotropic glutamate receptors. J Physiol 591(16):3935–3947
Luccini E et al (2007) Functional interactions between presynaptic NMDA receptors and metabotropic glutamate receptors co-expressed on rat and human noradrenergic terminals. Br J Pharmacol 151(7):1087–1094
Tu JC et al (1999) Coupling of mGluR/Homer and PSD-95 complexes by the shank family of postsynaptic density proteins. Neuron 23(3):583–592
Blümcke I et al (1996) Immunohistochemical distribution of metabotropic glutamate receptor subtypes mGluR1b, mGluR2/3, mGluR4a and mGluR5 in human hippocampus. Brain Res 736(1–2):217–226
Nomura A et al (1994) Developmentally regulated postsynaptic localization of a metabotropic glutamate receptor in rat rod bipolar cells. Cell 77(3):361–369
Sortino MA et al (1996) Immortalized hypothalamic neurons express metabotropic glutamate receptors positively coupled to cyclic AMP formation. Eur J Neurosci 8(11):2407–2415
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Leyrer-Jackson, J.M., Namba, M.D., Hood, L.E., Olive, M.F. (2021). Using Western Blotting for the Evaluation of Metabotropic Glutamate Receptors Within the Nervous System: Procedures and Technical Considerations. In: Olive, M.F., Burrows, B.T., Leyrer-Jackson, J.M. (eds) Metabotropic Glutamate Receptor Technologies. Neuromethods, vol 164. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1107-4_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1107-4_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1106-7
Online ISBN: 978-1-0716-1107-4
eBook Packages: Springer Protocols