Trafficking & Internalization

Trafficking of Receptors, Transporters and Ion Channels

A More Complete Picture

Receptor Activation, Ion Transport, and Transporter Translocation; all important processes for the largest class of Pharma targets. But, each of these targets shows another mode of action that is just now becoming appreciated – their trafficking to the cell surface, their internalization upon activation, and their eventual degradation or recycling.

The affect of compounds on these independent processes can be as profound as the “classical” mode of pharmacology for these surface proteins.

Comprehensive Trafficking Assays at Sharp Edge Labs

With technology licensed exclusively from Carnegie Mellon University, we’re able to dissect each step in the life-cycle of a surface protein to better understand the trafficking of the target, and defects in trafficking caused by mutation.

Using our patented Fluorogen Activating Module technology, and the suite of Fluorogens , these assays allow the unique identification of the components of the trafficking cycle.

  1. Cell Surface Protein

Selective Surface labelingBecause se-Red-xc selectively labels the plasma membrane protein fraction, you can easily quantify the proteins that’s been successfully trafficked to the surface, as well as providing clear definition of the cellular envelope for high-content measurements.  No washing is required for this selective labeling.

  1. Total Protein

Here we see that unlike se-Red-xc, which labels only surface protein, se-Red-s labels total protein (which includes surface protein, and protein being processed in the Golgi/ER complex for export to the plasma membrane).  Note that these fluorogen can be used interchangeably, you can add se-Red-xc first (for surface protein) followed by se-Red-s to quantify the additional intracellular protein. Again, no washing is required following addition of these flurogenic dyes.

  1. Endocytic Fraction and Recycling,

Here we use the pH sensor fluorogen, se-Red-pH, to independently track surface protein (the high pH fraction) and the endosomal fraction (the low pH fraction).  The video shows the live-cell experiment.  The figure below shows the data analysis showing the behavior of these two distinct protein populations over time.

A similar set of studies can be performed to track receptor recycling.  In this case we remove the antigen and the low pH fraction is recycled to the surface where the pH returns to normal.  Note that the lower signal after recycyling is an indication of the extent of lysosomal degradation.

Available as Custom Assay Service or as Reagents

Contact Us for more information on generating this data for your target protein of interest.

*The FAPs are covered by US Patent 8,664,364 and other US and international pending patent applications.