Abstract
Nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, has the potential to quantitatively characterize protein post-translational modifications that cause shifts in isoelectric point (pI). This protocol details the NIA analysis of protein phosphorylation using AKT as an example. This protocol can be used for two platforms, NanoPro 1000 and Peggy Sue, from ProteinSimple Company. NanoPro 1000 separates proteins based on charge. Peggy Sue separates proteins based on either charge or size. The platforms can analyze up to 96 samples at a time with lysates from as few as 25 cells per assay. Detailed information of the platforms is available on ProteinSimple’s website (http://www.proteinsimple.com).
Keywords: Immunoassay, Antibody, AKT, NIA, Phosphorylation
Materials and Reagents
Equipment
Software
Procedure
Representative data
Notes
Recipes
Acknowledgments
The work was supported by National Institutes of Health (NIH) grant 5R21CA126700 and a grant from the University of Texas MD Anderson Cancer Center Kidney Cancer Multidisciplinary Research Program to ZD. The protocol was adapted from our published paper in Oncogene (Guo et al., 2014) with modifications.
References
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