NanoPro Applications
- Quantify phosphoprotein isoforms in cells and tissues
- Measure signaling proteins in sorted stem cells and primary cells
- Monitor drug actions in tissue sections and fine needle aspirates
NanoPro Systems address a critical need among researchers of cancer, diabetes, and many other diseases by providing a unique window into cell signaling processes within tiny biological samples. While traditional analytical techniques such as Western blots and mass spectrometry can consume 100,000 or more cells per measurement, NanoPro assays can be run with as few as 25 cells. This capability enables protein analysis in many sample types previously considered much too small for protein analysis.
Applications :: Quantitation of Phosphoprotein Isoforms in Cell Lines
NanoPro's high-resolution IEF capillary separation provides outstanding resolution of phosphoprotein isoforms. By using a pan-specific antibody to recognize all non-phosphorylated and phosphorylated isoforms, relative phosphorylation levels can be estimated. In the example shown below, MCF10A breast cancer cells were serum starved (control) and then stimulated with 10 ng/mL EGF for 25 minutes (stimulated). Simple area-under-the-curve calculations show that the proportion of phosphorylated MEK1 increased from 70% to 92% after stimulation.
MEK1 in breast cancer cells
Applications :: Signaling Proteins in Sorted Cells
Fluorescence-activated cell sorting (FACS) is routinely used to isolate pure cell populations from complex biological tissues. Small populations of sorted cells can be analyzed on NanoPro systems because of their high detection sensitivity. One simple example is shown in the figure below. Peripheral blood leukocytes from a single mouse were stimulated for 24 hours with anti-CD3 and anti-CD28. The stimulated cells were sorted into populations of B cells (A) and T cells (B), then assayed for stimulation of ERK1 and ERK2. As expected, ERK1 and ERK2 phosphorylation increased markedly in the T cells, but not in the B cell population.
ERK1 and ERK2 stimulation in B cells vs T cells from a single mouse
Applications :: Drug Actions in Xenograft Fine Needle Aspirates
Fine needle aspirates (FNAs) taken from mouse xenografts typically contain too few cells for traditional protein analysis. However, these samples provide ample material for NanoPro analysis. In fact, investigators from Stanford University have used FNA analysis on the NanoPro 1000 to monitor drug effects in individual mice over time. The figure below shows data generated from a tumor xenograft formed following subcutaneous injection of leukemia cells into a SCID mouse. The Gleevec-induced decrease in STAT5 phosphorylation was dramatic, while the level of HSP70 loading control remained essentially constant.
Gleevec decreases STAT5 phosphorylation in mouse xenografts of leukemia cells.
