Prostanoids are the cyclooxygenase metabolites derived from C-20 unsaturated fatty acids (arachidonic acid) and include prostaglandin (PG) D2, PGE2, PGF2 alpha, PGI2, and thrombaxane (Tx) A2. The fatty acids precursors are released from the membrane phosphoplipids in response to various physiological and pathological stimuli by the action of phospholipase A2 and are converted to various prostanoids by the sequential actions of cyclooxygenases and the respective synthases. Prostaglandin PGE2 is one of the major prostaglandin produced during inflammation. A variety of PGE2-mediated effects on vascular smooth muscle tonus, glomerular cell function, renin release, and renal salt and water transport have been described. PGs also influence neuronal activity by modulating neurotransmitter release, sensitizing secretory fibers to noxious stimuli, or inducing fever and sleep. The actions of PGE2 are mediated by rhodopsin-type; G-protein coupled membrane receptors, termed E-prostanoid (EP) receptors or PTGERs. There are four subtypes of PGE receptors designated as EP1, EP2, EP3, and EP4 that are encoded by different genes and expressed differently in each tissue. The intracellular signaling also differs among the receptor subtypes. In general, EP receptors display a protein topology typical of GPCR-7 TM domain, an extracellular N-terminus, and a large intracellular C-terminus.
EP4 (mouse 513 aa, rat 488 aa; human 488 aa, chromosome 5p13.1, ~ 90% interspecies homology) is expressed in intestine, lung, thymus, kidney, uterus, pancreas, spleen, heart, stomach, and brain. EP4 knockout mice show patent ductus arteriosus, and decreased inflammatory bone resorption. EP4 activity is mediated by Gs that stimulate adenyl cyclase.It has a relaxing effect on smooth muscle and implicated in renal hemodynamics, intestinal epithelial transport, adrenal aldosterone secretion, and uterine functions. It was originally reported as EP2 subtype.
An 18aa peptide within C-terminal, cytoplasmic domain # 4 of Human EP4 (1) was coupled to KLH and antibodies produced in rabbits. Antibodies were affinity purified using the control peptides linked to Sepharose.
Applications
Suitable for use in ELISA, Western Blotting. Other applications not tested.
Western Blot: 1:1000-1:5000 for neat serum and 1-10ug/ml for affinity pure antibody using ECL. EP4 has been shown to be 47 and 78kD EP4 bands in the kidney (2).
ELISA: 1:10,000-50,000 for neat serum and 0.5-1ug/ml for affinity pure. Control peptide can be used to coat ELISA plates at 1ug/ml. Optimal dilutions to be determined by the researcher.
Storage and Stability
May be stored at 4°C for short-term only. For long-term storage and to avoid repeated freezing and thawing, aliquot and add glycerol (40-50%). Freeze at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Immunogen
An 18 AA peptide within C-terminal, cytoplasmic domain 4 of Human EP4 (1) coupled to KLH
Form
Supplied as a liquid, neat serum, 40% glycerol.
Purity
Purified using the control peptides linked to Sepharose
Specificity
The human control peptide is 100% conserved in rabbit, 87% in canine, 94% in rat and mouse EP4. No significant homology exist with other EP (1, 2 or 3) receptors. Antibody crossreactivity in various species is not established. The Control peptide, because of the small size (~3kD), is not suitable for Western. It should be used in ELISA or to neutralize antibody and confirm specificity of antibody. (use 5-10 ug of peptide per 1 ug of aff pure or 1 ul of antiserum) to demonstrate antibody specificity.
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