Determination of Phosphorylation Sites in Microtubule Associated Protein MAP65-1

2014 ◽  
pp. 161-170 ◽  
Author(s):  
Andrei Smertenko
Keyword(s):  
Phosphorylation Sites

scholarly journals Determination of the tyrosine phosphorylation sites in the T cell transmembrane glycoprotein CD5

2001 ◽  
Vol 13 (2) ◽  
pp. 149-156 ◽  
Author(s):  
Kevin M. Dennehy ◽  
William F. Ferris ◽  
Hanne Veenstra ◽  
Linda A. Zuckerman ◽  
Nigel Killeen ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Phosphorylation Sites ◽  
Transmembrane Glycoprotein

scholarly journals High specificity of widely used phospho-tau antibodies validated using a quantitative whole-cell based assay

2019 ◽  
Author(s):  
Dan Li ◽  
Yong Ku Cho
Keyword(s):  
Specific Binding ◽  
High Specificity ◽  
Tau Proteins ◽  
Phosphorylation Sites ◽  
Human Embryonic Kidney ◽  
Post Translational Modification ◽  
Flow Cytometry Assay ◽  
Hek Cells ◽  
Mouse Hippocampus

AbstractAntibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies. In order to quantify and compare the specificity phospho-tau antibodies and other post-translational modification site-specific antibodies in general, a measure of specificity is urgently needed. Here we report a robust flow cytometry assay using human embryonic kidney (HEK) cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non-specific signal in antibody binding. We validate our assay using anti-tau antibodies with known specificity profiles, and apply it to measure the specificity of 7 widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have Φ values near 1, which indicates no detectable non-specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non-specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non-specific binding of AT270 to non-tau proteins found in HEK cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general.

Determination of the Phosphorylation Sites of Calmodulin Catalyzed by Casein Kinase 21

1988 ◽  
Vol 104 (6) ◽  
pp. 946-951 ◽  
Author(s):  
Shigeo Nakajo ◽  
Yutaka Masuda ◽  
Kazuyasu Nakaya ◽  
Yasuharu Nakamura
Keyword(s):  
Casein Kinase ◽  
Phosphorylation Sites

scholarly journals Facile Determination of Phosphorylation Sites in Peptides Using Two-Dimensional Mass Spectrometry

2020 ◽  
Vol 92 (10) ◽  
pp. 6817-6821
Author(s):  
Johanna Paris ◽  
Tomos E. Morgan ◽  
Christopher A. Wootton ◽  
Mark P. Barrow ◽  
John O’Hara ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Phosphorylation Sites ◽  
Two Dimensional

scholarly journals Identification of an IκBα-associated Protein Kinase in a Human Monocytic Cell Line and Determination of Its Phosphorylation Sites on IκBα

1995 ◽  
Vol 270 (46) ◽  
pp. 27914-27919 ◽  
Author(s):  
Kouji Kuno ◽  
Yuji Ishikawa ◽  
Mary K. Ernst ◽  
Masafumi Ogata ◽  
Nancy R. Rice ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Line ◽  
Phosphorylation Sites ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Human Monocytic Cell Line ◽  
Human Monocytic Cell
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