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dc.contributor.authorLee, Byung Rho
dc.contributor.authorKamitani, Tetsu
dc.date.accessioned2012-10-26T20:28:39Z
dc.date.available2012-10-26T20:28:39Z
dc.date.issued2011-08-19en_US
dc.identifier.citationPLoS One. 2011 Aug 19; 6(8):e23939en_US
dc.identifier.issn1932-6203en_US
dc.identifier.pmid21886844en_US
dc.identifier.doi10.1371/journal.pone.0023939en_US
dc.identifier.urihttp://hdl.handle.net/10675.2/754
dc.description.abstractα-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinson's disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.
dc.rightsLee, Kamitani. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_US
dc.subjectResearch Articleen_US
dc.subjectBiologyen_US
dc.subjectBiochemistryen_US
dc.subjectImmunochemistryen_US
dc.subjectProteinsen_US
dc.subjectImmunologyen_US
dc.subjectImmunologic Techniquesen_US
dc.subjectImmunoassaysen_US
dc.subjectNeuroscienceen_US
dc.subjectMolecular Neuroscienceen_US
dc.subjectMotor Systemsen_US
dc.subjectMedicineen_US
dc.subjectClinical Immunologyen_US
dc.subjectImmunologic Techniquesen_US
dc.subjectImmunoassaysen_US
dc.subjectNeurologyen_US
dc.subjectParkinson Diseaseen_US
dc.titleImproved Immunodetection of Endogenous α-Synucleinen_US
dc.typeArticleen_US
dc.identifier.pmcidPMC3158774en_US
dc.contributor.corporatenameDepartment of Medicine
dc.contributor.corporatenameCenter for Molecular Chaperone/Radiobiology & Cancer Virology
refterms.dateFOA2019-04-10T00:47:07Z
html.description.abstractα-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinson's disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.


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