In the assay, the receptor can be epitope-tagged and an antibody recognizing the tag is then labelled with CypHer5. Alternatively, an endogenous antibody that recognizes an exofacial epitope can be labelled directly with CypHer5. Agonist-induced internalization of the receptor is then measured as an increase in fluorescence in the cell. This offers an advantage over other techniques that rely solely on measuring a translocation in fluorescent signal. Many other commonly used techniques such as b-arrestin recruitment, Ca2+ flux, and cAMP measurement are only suitable for use with G-protein coupled receptors (GPCRs). In contrast, CypHer5 is functional with both GPCRs and non-GPCRs that internalize in response to external stimuli.
GPCRs represent an important area for target validation. To demonstrate the generic applicability of CypHer5 pH-sensitive dye, examples of the GPCR subfamilies Gi (δ -opioid receptor), Gq (TRHR-1, angiotensin II) and Gs (β -2 adrenergic receptor) (3) were studied. These assays involve the use of CypHer5 labelled anti-VSV-G antibody to detect the VSV-G tagged receptors.
Epidermal growth factor receptor (EGFR) plays a central role in the complex signaling pathways involved in cell growth and differentiation (4) and is an example of a non-GPCR that is widely studied. The assay described here uses an exofacial antibody for EGFR that is directly labelled with CypHer5 Mono NHS Ester.
CypHer5 labelled anti-VSV-G antibody, 250 mg PA45407