Detection of Rat and Mouse GPR64 by Western Blot.
Western blot shows lysates of rat epididymis tissue and mouse epididymis tissue. PVDF membrane was probed with 2 µg/mL of Sheep Anti-Human/Mouse/Rat GPR64 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7977) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for GPR64 at approximately 150 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
GPR64 in Human Epididymus.
GPR64 was detected in immersion fixed paraffin-embedded sections of human epididymus using Sheep Anti-Human/Mouse/Rat GPR64 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7977) at 3 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific staining was localized to plasma membranes of epithelial cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.
Detection of GPR64 in HEK293 Human Cell Line Transfected with Human GPR64 and eGFP by Flow Cytometry.
HEK293 human embryonic kidney cell line transfected with (A) Human GPR64 or (B) Human GPR75 and eGFP was stained with Sheep Anti-Human GPR64 Affinity-Purified Polyclonal Antibody (Catalog # AF7977) followed by Allophycocyanin-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # F0127). Quadrant markers were set based on control antibody staining (Catalog # 5-001-A). View our protocol for Staining Membrane-associated Proteins.
Preparation and Storage
Sterile PBS to a final concentration of 0.2 mg/mL.
Reconstitution Buffer Available
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
Stability & Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.
GPR64 (G-Protein coupled Receptor 64; also HE6) is a 110 kDa (predicted) member of the B class of GCPRs. Within this class GPR64 belongs to a Large N-termini family-B 7-transmembrane (LNB-7TM) subclass of receptors (also known as adhesion-GCPRs). GPR64 has restricted expression, being found in stereocilia cell membranes of epididymal caput epithelial cells and, to a limited extent, on osteoblasts. The function of GPR64 is somewhat unclear, but in the epididymis, it may be involved in fluid transport. Mature human GPR64 is a 980 amino acid (aa) 7-TM glycoprotein (SwissProt Q8IZP9). It contains an extended extracellular N-teminus (aa 38-627), seven consecutive TM segments (aa 628-878) and a C-terminal cytoplasmic tail (aa 879-1010). The extended extracellular region possesses a juxtamembrane GPS domain (aa 567-618) that serves as a proteolytic cleavage site. Enzymatic activity here generates a 180 kDa soluble form that stays associated with a 40 kDa membrane-embedded fragment. Notably, isolation of the membrane fragment gives rise to oligomers that run at > 250 kDa in SDS-PAGE. There are multiple splice variants. The one used for immunization to generate this antibody contains a deletion of aa 88-101 (RefSeq NP_001073328). Four other splice forms show single block deletions of aa 65-67, 51-66, 52-75, and 906-956, respectively. Three others possesses aa substitutions; a 20 aa block for aa 52-101, and a common 12 aa block that can substitute for either aa 68-101 or aa 52-101. Over aa 38-64 and 68-553 of RefSeq NP_001073328, human and mouse GPR64 share 69% aa sequence identity.
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