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The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR


Introduction
CypHer5TM is a red-excited, pH-sensitive cyanine dye that is non-fluorescent at pH 7.4 and maximally fluorescent at pH 5.5. It is therefore ideally suited for monitoring internalization of cell surface receptors via acidic endosomal vesicles upon agonist stimulation (1, 2).

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.


Products used
CypHer5 labelled anti-VSV-G antibody, 250 mg PA45407

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