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PathHunter® Protein Translocation Assays

Track Cellular Movement of Proteins to Multiple Membrane Compartments

The movement of proteins from the endoplasmic reticulum to plasma membrane, cytoplasm to nucleus, or from plasma membrane to endosomes is essential for their specific biological role and the function of the cell. If these processes are altered due to protein mutations or abnormal signaling, undesirable effects related to their cellular movement may occur, often resulting in diseases including cystic fibrosis, Alzheimer’s disease, and Huntington’s disease. PathHunter protein translocation cell-based assays provide the ability to study trafficking of your protein of interest to and from these different cellular compartments, and analyze the pharmacological effects of compounds on these translocations.

 
Protein Translocation Image
PathHunter protein translocation assays are based on the proprietary Enzyme Fragment Complementation (EFC) technology and provide a tool for trafficking of proteins into different cellular membrane compartments (nucleus, cell membrane, or endosome). PathHunter cells are genetically engineered to over-express the translocating protein under study fused to a small fragment of β-galactosidase (β-gal) called the enzyme donor, ED [ProLabel® (PL) or ProLink™ (PK)], and a larger fragment of β-gal called enzyme acceptor (EA) that is localized to the nucleus, cell membrane, or endosome. Upon induction of cells (e.g. ligand stimulation), the PL or PK-tagged protein undergoes translocation to the cellular compartment in which the EA-is localized. High affinity complementation between the two fragment, PL/PK and EA, results in the formation of an active β-gal enzyme that cleaves a substrate to generate chemiluminescent signal.

Explore Protein Translocation Assay Platforms

PathHunter GPCR Internalization Assays are functional assays that offer a unique and scalable method to monitor the internalization and recycling patterns of activated GPCRs

PathHunter Pharmacochaperone Trafficking Assays for broad pharmacological characterization and interrogation of compound function in disease processes associated with membrane protein (e.g. GPCRs, ion channels, transporters) trafficking and internalization due to protein misfolding.

PathHunter Signaling Pathway Protein Translocation Assays for identification and confirmation of specific activation or inhibition of particular signaling pathways.

PathHunter Nuclear Hormone Receptor Translocation Assays

These assays detect binding of an agonist or antagonist to the ligand binding domain of the nuclear hormone receptor (NHR) of interest and triggering of NHR activation and translocation into the nucleus.

Nuclear Translocation Principle
 

PathHunter Receptor Tyrosine Kinase (RTK) Internalization Assays

These assays monitor the internalization of wild type, full length RTKs in an antibody-free, homogeneous (no wash) HTS-friendly assay format.

Receptor Tyrosine Kinase Internalization Assay
 
DiscoverX offers a variety of products and services for tracking the movement of proteins to multiple membrane compartments.


PathHunter GPCR Internalization Assays

PathHunter Signaling Pathway Protein Translocation Assays

PathHunter Nuclear Hormone Receptor Translocation Assays

PathHunter Receptor Tyrosine Kinase Internalization Assays


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Utilize our Custom Assay Development services to create custom constructs, cell pools, or clones in a variety of cell types.

Protein Translocation Assays Highlights

  • Explore protein movement using a non-imaging, high throughput cell-based assay platform
  • Study pharmacology of ligands that trigger protein translocation to the plasma membrane, endosome, or nucleus
  • Create your own quantitative cell-based assays to study translocation of any protein
 

Make Your Own Cell-Based Protein Translocation Assays

Study trafficking or translocation of proteins from one cellular compartment to another.
Protein Translocation Assay Principle

Generate a PathHunter translocation assay by first creating a plasmid vector with your target protein of choice tagged with enzyme donor [ED; ProLink (PK) or enhanced ProLabel® (ePL)]. Simply transfect this plasmid into a PathHunter EA parental cell line containing an EA-reporter protein (e.g. Endosome-EA [ENDO-EA] as shown in this example), and then perform a translocation assay  in the presence of a ligand. GPCR “total”-internalization from the cell membrane to the endosome is shown.

For creating a trafficking assay from the endoplasmic reticulum to the plasma membrane use the Membrane-EA [MEM-EA] PathHunter EA parental cell line (see customer publication).
 


Analyze Membrane Protein Internalization to the Endosome

Measure protein internalization of membrane proteins like GPCRs or receptor tyrosine kinases (RTKs) from the plasma membrane to the endosome upon ligand stimulation.

OPRM1 Total GPCR Internalization Cell Line
A GPCR internalization assay was made using ENDO-EA U2OS parental cells expressing PK1-tagged opioid receptor mu, OPRM1 (Cat. No. 93-0745C3). The data indicate translocation of the ED-tagged receptor from the plasma membrane to the EA-tagged endosome upon stimulation with agonist [Met5]- enkephalin (EC50 = 589 nM; signal/background (S/B) = 4.3).
 


Quantify Nuclear Protein Translocation to the Nucleus

Obtain quantitative data showing binding and ligand induced translocation of nuclear hormone receptors (NHRs) such as androgen, glucocorticoid, liver X, mineralcorticoid, and progesterone receptors.

CHO-K1 Glucocorticoid Receptor Nuclear Translocation Cell Line
A nuclear translocation assay was generated using NUC-EA CHO-K1 parental cells stably transfected with PL-tagged glucocorticoid receptor (Cat. No. 93-0002C2). The data indicate successful translocation from the cytoplasm to the nucleus, where EA is localized, upon stimulation of ligand dexamethasone (EC50 = 8.2 nM; S/B = 15.9).
 


Discover Pharmacochaperones That Rescue Disease-Associated Mutant Receptors

Identify pharmacochaperones compounds which function by promoting proper folding of misfolded GPCRs or ion channels and assist in trafficking from the endoplasmic reticulum (ER) to the plasma membrane.  
 


KCNH2(G601S) Pharmacotrafficking Assay

Mutations in the potassium voltage-gated channel human ERG (KCNH2), can lead to reduced functional potassium current, long QT syndrome, and cardiac arrhythmias. A pharmacotrafficking assay was created using MEM-EA U2OS parental cells stably transfected with PK-tagged mutant KCNH2(G601S) (Cat. No. 93-1064C3). The pharmacochaperone clofilium was able to rescue the mutant ion channel by promoting proper folding resulting in successful trafficking from the ER to the membrane (EC50 = 564 nM; S/B = 3.5).
 


Easily Investigate Multi-Subunit ion Channel Protein Assembly

 

Calcium Ion Channel Subunit Assembly
A voltage-dependent calcium channel (Cav2.2) subunit assembly assay was created using MEM-EA parental cells stably transfected with PK-tagged subunit A1B (one of the three Cav2.2 subunits), along with neither, one, or both of the untagged Cav2.2 partner subunits 2D1 and B3. Low basal levels of EFC activity are obtained when only one or two subunits are present, indicating little to no trafficking from the ER to the plasma membrane. Only by adding all three subunits, A1B-PK/2D1/B3, Cav2.2 forms a proper ion channel subunit assembly and trafficking achieved.