ProteinSimple's NanoPro 1000, the nanofluidic immunoassay platform used in the Proteomics Core, employs separation of native proteins by isoelectric focusing (IEF) in small capillaries (400 nanoliter volumes). Once focusing is complete, proteins are cross-linked to the walls of the capillaries and immunoassay reagents — such as primary, horseradish peroxidase (HRP)-labeled secondary antibody or hydrogen peroxide-luminol — are introduced.
This assay is very sensitive to low-abundance antigens, and because proteins are separated based on charge, phosphoproteins are resolved from nonphosphorylated forms, allowing quantitation of peak areas of the different isoforms.
Assays for several major cell signaling proteins and their phosphorylated forms are available, including ERK1/2 (MAPK), Akt, EGFR, MEK1 and MEK2. All NanoPro assay projects must be reviewed and approved by the Proteomics Core.
- Considerations. Antibody selection is critical, as most commercial antibodies that work for traditional Western blots will not recognize the native protein in this assay.
Process. ProteinSimple's NanoPro 1000 is used for the assay. Cell culture lysates, tissues or cells derived from fine-needle aspirates, primary cells or cells sorted by florescence-activated cell sorting are prepared in low-salt lysis buffer. Small volumes of lysate (10 to 20 microliters) at protein concentrations of 20 to 200 micrograms per milliliter are pipetted into 384-well microtiter plates. Other reagents also are added to additional rows.
Up to 96 samples can be assayed in one overnight run. At the end of the IEF step, the signals from the fluorescently labeled ampholyte standards are captured by a charge-coupled device (CCD) camera and compared with the chemiluminescent signal to align the peaks from different samples.
- Reporting. Results can be viewed graphically, including the creation of virtual gel images, using ProteinSimple software, or the data can be exported into spreadsheet format for analysis.
Project costs are on a per-capillary basis, along with applicable technician time for sample preparation and analysis.
For more information or to initiate a service or project request, email the Proteomics Core.