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|Human Cell lysate that Human ECE-2 transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|A DNA sequence encoding the ectodomain of human endothelin converting enzyme 2 isoform A (NP_055508.3) (Gly 199-Trp 883) was fused with a polyhistidine tag at the N-terminus.|
|The recombinant human ECE2 consists of 700 amino acids with the predicted molecular mass of 80.2 kDa. As a result of glycosylation, rhECE2 migrates as an approximately 120-125 kDa band in SDS-PAGE under reducing conditions.|
|Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.|
|Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt.|
|1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
Endothelin-converting enzyme 2, also known as ECE-2, is a metalloprotease that possesses many properties consistent with it being a neuropeptide-processing enzyme. Endothelin-converting enzymes (ECEs) are the key enzymes in the endothelin (ET) biosynthesis that catalyze the conversion of big ET, the biologically inactive precursor of mature ET. Two enzymes, termed ECE-1 and ECE-2, have been molecularly identified. ECE-2 is found primarily in neural tissues, with high levels of expression in midbrain, cerebellum, hypothalamus, frontal cortex and spinal cord and moderate levels in hippocampus and striatum. ECE-2 is strongly down-regulated in inferior parietal lobe from Alzheimer disease patients (at protein level). ECE-2 converts big endothelin-1 to endothelin-1. It is involved in the processing of various neuroendocrine peptides, including neurotensin, angiotensin I, substance P, proenkephalin-derived peptides, and prodynorphin-derived peptides. ECE-2 may limit beta-amyloid peptide accumulation in brain. It may also have methyltransferase activity. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1.