Translational Applications Service Center

NIA and SimpleWestern Overview

TASC offers its users two state of the art nano-fluidic immunoassay technologies from ProteinSimple (charge-separation based nano-immunoassay, and size-separation based nano-immunoassay: SimpleWestern) for defining protein signatures and measuring proteomic response to targeted therapies in rare clinical specimens. These novel proteomic technologies are currently unavailable elsewhere at Stanford, and there are no commercial providers that can offer this assay service.

TASC has recently acquired the newest ProteinSimple assay platform, PeggySue, capable of separating proteins based on both charge (NIA), as well as size (quantitative capillary-based SimpleWestern).  Using NIA and SimpleWestern technology to observe changes in various signaling pathways, new assays developed at TASC have helped define various novel targets for cancer, heart disease, pulmonary hypertension, and several other diseases.

Nano Immuno-Assay is an automated nano-fluidic method that uses isoelectric protein focusing and antibody detection to quantify protein expression and resolve un-phosphorylated from single- and multiple- phosphorylated protein isoforms in as few as 25 cells of starting material. All steps are performed in a single capillary. A precision robot moves capillaries to the sample, reagent, incubation, and separation chambers as dictated by the assay protocol. To date, assays have been developed for over 20 proteins to observe proteomic changes in proliferation, cell cycle, apoptosis and various signaling pathways.

O'Neill, R.A. et al. Isoelectric focusing technology quantifies protein signaling in 25 cells. Proc. Natl. Acad. Sci. USA 103, 1615316158 (2006).

Fan AC, et al. Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens. Nat Med. 2009 May;15(5):566-71.

Should You Use Charge (NIA) or Size (SimpleWestern) Assay for your Project?

 

How NIA (charge-based) Assays Work

Load
The capillary is filled with a 400-nL mixture of sample, fluorescently labeledo pI standards and ampholytes
Separate
Voltage is applied across the capillary to drive the IEF or MW separation. Individual proteins and pI standards concentrate at their isoelectric points, and the position of each standard in the capillary is recorded.
Immobilize
The capillary is exposed to UV light, activating the proprietary linking chemistry and locking the separated protein isoforms to the capillary wall.
Immunoprobe
The capillary is rinsed and immunoprobed for specific proteins. Luminol and peroxide are added to generate chemiluminescent light, which is captured by a CCD camera.
Quantitate
The digital image is analyzed and quantitative results are presented in the software.

How SimpleWesterns (size-based) Assays Work

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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