Protein Translocation Assays

Track Cellular Movement of Proteins to Multiple Membrane Compartments

The movement of proteins from the plasma membrane to endosomes, endoplasmic reticulum to plasma membrane, or cytoplasm to nucleus is essential for their specific biological role and the function of the cell. If these movements are altered by therapeutic binding, protein mutations, or abnormal signaling; undesirable consequences may occur such as drug tolerance, unwanted side effects, therapeutic toxicity, and diseases. Measuring therapeutic binding that alters translocating proteins provides insight into these adverse effects and ultimately helps with identifying safer drugs.

PathHunter® protein translocation assays provide the ability to study internalization and trafficking of your protein of interest to and from different cellular compartments, and analyze the pharmacological effects of therapeutics on these events. Unlike alternative methods that require specialized equipment, antibodies, or fluorescent tags, these cell-based assays are high throughput, easy-to-use, quantitative, and highly specific and sensitive.

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Advantages of Translocation Assays

  • Simple, Rapid Assay – Explore protein movement to multiple cellular compartments using an easy-to-use, non-imaging, high throughput cell-based assay platform
  • Flexible Solutions – Create your own quantitative cell-based assays to study translocation of any protein (e.g. GPCRs, RTK, checkpoint receptors, nuclear proteins, ion channels, & more)
  • Pharmacochaperone Discovery – Identify small molecule compounds that functionally rescue disease relevant mutant proteins involved in cystic fibrosis, Alzheimer’s disease, Huntington’s disease, and others
  • Immuno-Oncology Application – Quantify antibody-based therapeutics targeting IO receptors that can induce high levels of internalization and serve as candidates for conjugation to cytotoxic compounds

Key Resources

Eurofins DiscoverX offers a variety of cell-based assays for tracking the movement of proteins to multiple membrane compartments.

Internalization Assays for GPCRs, Checkpoint & Cytokine Receptors, and Receptor Tyrosine Kinases (RTKs)


Pharmacotrafficking Assays for GPCRs, Ion Channels, and Transporters


Translocation Assays for Signaling Pathway Nuclear Proteins and Nuclear Hormone Receptor (NHR)


Create Your Own Cell-Based Translocation 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.
PathHunter protein translocation assays are based on the proprietary Enzyme Fragment Complementation (EFC) technology to measure the movement of proteins into different cellular membrane compartments (e.g. to the cell membrane, nucleus, or endosome).

Internalization Assays for GPCRs, Checkpoint Receptors and Receptor Tyrosine Kinases (RTKs)

These assays monitor the internalization of membrane receptors in an imaging and antibody-free, homogeneous (no wash) HTS-friendly assay format. For GPCRs, there is an alternative format to the assay principle shown below for measuring activated internalization through the GPCR-β-arrestin complex. Refer to GPCR Internalization Assays for details.


PathHunter Receptor Internalization Assays detect membrane receptor translocation to the endosome. Cell lines are engineered to co-express the ProLink™ (PK; EFC enzyme donor)-tagged membrane receptors, and an EFC enzyme acceptor (EA) tag localized to the endosomes. Internalization assays for both multi-pass membrane receptors (GPCRs) and single-pass membrane receptors (RTKs or immune checkpoint receptors) have been developed for this assay platform. Small molecules or biologics (e.g. antibodies) that induce activation of the receptor-PK fusion protein leads to internalization of the receptor to the EA-tagged endosomes, forcing complementation of the two β-galactosidase (β-gal) enzyme fragments (EA and PK). The resulting functional β-gal enzyme hydrolyzes a substrate to generate a chemiluminescent signal.

Pharmacotrafficking Assays for GPCRs, Ion Channels, and Trasporters

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

Translocation Assays for Signaling Pathway Nuclear Proteins

Signaling Pathway Nuclear Proteins assays allow for the identification, screening, lead optimization, and even safety testing of therapeutics.  These assays monitor the activation or inhibition of a particular signaling pathway coupled with the movement of proteins from the cytosolic compartment to the nuclear compartment. In general, this approach enables you to create a highly specific, non-transcriptional assay for almost any protein that is known to translocate to the nucleus, such as FOXO3, NRF2, XBP1, p53 and many others.


Elucidation of NRF2 Translocation using PathHunter Protein Translocation Assay Platform. NRF2 [Nuclear factor (erythroid-derived 2)-like 2], a transcription factor, and its downstream target genes play an important role in cellular anti-oxidant defense. In this assay, the target protein is tagged with the EFC enzyme donor (ED), and the enzyme acceptor (EA) is confined to the nuclear compartment, using a nuclear localization tag. When the target protein, such as a transcription factor is activated and it translocates to the nucleus, it brings the ED and EA into close proximity and creates an active β-gal enzyme. This active enzyme is able to hydrolyze the substrate and create chemiluminescent light, detectable on any plate-reader.

Translocation Assays for Nuclear Hormone Receptor (NHR)

NHR translocation assays detect binding of a small molecule agonist or antagonist to the ligand binding domain of an NHR triggering its activation and translocation into the nucleus.


General assay principle of PathHunter Nuclear Translocation assays. The cells have been engineered to express the ED fragment fused to the NHR, and EA fragment localized in the nucleus. Activation of the NHR induces receptor translocation to the nucleus, forcing complementation of the ED and EA fragments. Activity of the resulting fully formed β-galactosidase enzyme is detected using a chemiluminescent substrate.

There are several applications for translocation assays. Select applications shown below. For additional applications, please refer to the GPCR Internalization, Pharmacotrafficking and Toolbox pages.

Profile Molecules That Trigger Receptor Internalization and Compare Results to Other Assay Types


Data from the PathHunter GPCR CCKAR (cholecystokin A receptor) Internalization Assay can be used for comparison to results from other GPCR CCKAR Assays (β-arrestin recruitment, cAMP accumulation, or calcium flux) evaluating receptor signal activity in response to the same ligands. Combining knowledge of how ligand/compound binding affects receptor signaling events, as well as receptor localization and internalization, can provide insights into the overall effects on efficacy and safety.


Four human δ-Opioid receptor (hDOR) agonists were characterized for their activation of receptor internalization and the inhibition of forskolin-stimulated cAMP accumulation. Cells overexpressing the hDOR receptor in the PathHunter Activated GPCR Internalization [A.] and cAMP Hunter [B.] formats were treated with known agonists and assayed using PathHunter and HitHunter® Detection reagents respectively. For comparison, the data was normalized to [Met5]-enkephalin in potency (value set to 1) and efficacy (set to 100%). Despite similar compound potencies and efficacies based on the inhibition of cAMP accumulation in the cAMP assay, SNC-80, which is known as a strongly internalizing compound and functional antagonist is clearly defined as a super-agonist in the internalization assay. The super-agonist effect suggests that SNC-80 may lead to higher levels of internalized receptor, and subsequently lower levels of receptor reserve – thus behaving as a “functional antagonist.”


Monitor RTK Internalization Through Ligand or Receptor Antibody Activation


PathHunter RTK Internalization Assays can be activated with ligands or receptor antibodies. [A.] Insulin receptor b (INSRb) internalization in response to incubation with increasing concentrations of insulin. [B.] Epidermal growth factor B2 (ErbB2) receptor internalization is detected when cells are incubated with two different ErbB2 antibodies including the therapeutic antibody trastuzumab (Herceptin®). Similar robust ErbB2 internalization was observed when cells were incubated with Herceptin and pertuzumab (Perjeta®; data not shown). Herceptin and Perjeta are registered trademarks of Genentech USA, Inc. Graphs’ x-axes use log-scale.

Study Antibody-Drug Conjugates (ADCs) – Analyze Antibodies That Activate Checkpoint Receptor Internalization


Checkpoint receptor antibodies activate receptor internalization. [A.] PathHunter internalization assay for the CD33 checkpoint receptor was tested with a commercial antibody to CD33. CD33 antibody was pre-incubated with a secondary antibody to cluster the CD33 receptor and then added to cells to monitor internalization. With higher cross-linked antibody concentrations, greater amounts of PK-tagged CD33 complemented EA-tagged protein localized to the endosome. A number of ADCs for CD33 are in clinical trials for the treatment of acute myeloid leukemia.1 [B.] B-cell maturation antigen (BCMA) receptor internalization was activated with a commercial BCMA antibody. Several BCMA ADCs are in development as a therapeutic for multiple myeloma. Graphs’ x-axes use log-scale.


Measure Activation and Translocation of NHRs from the Cytoplasm to the Nucleus

Obtain quantitative data showing binding and ligand-induced translocation oh NHRs such as androgen, glucocorticoid, liver X, mineralcorticoid, and progesterone receptors.


Monitoring Glucocorticoid Receptor Nuclear Translocation. [A.] Immunofluorescence analysis of dexamethasone induced Glucocorticoid Receptor translocation into the nucleus using PathHunter CHO-K1 Glucocorticoid Receptor Nuclear Translocation cells.  [B.] Translocation EFC-based assay (Cat. No. 93-0002C2) of the same cells stimulated with dexamethasone for 3 hours and results shown an EC50 of 8.2 nM and excellent signal-to-background of 15.9. PathHunter NHR translocation assays employ full length NHRs providing more biologically relevant data, and shorter assay times allowing for fewer off-target effects.