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|Human Cell lysate that Mouse CES5 / Carboxylesterase-5 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 extracellular domain of mouse CES5 (NP_766347.1) (Met 1-His 556) was expressed, with a C-terminal polyhistidine tag.|
|The secreted recombinant mouse CES5 comprises 541 amino acids and has a calculated molecular mass of 60.6 kDa. As a result of glycosylation, the recombinant protein migrates as an approximately 52 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.
Carboxylesterase belongs to the class of serine hydrolases family which includes Chymotrypsin and Acetylcholinesterase. Carboxylesterase is involved in the chemical reaction, exerting its role in catalyzing the carboxylic ester and water to convert to an alcohol and a carboxylate. Carboxylesterase is a type of enzyme that capable of hydrolyzing a variety of carboxylic acid esters and it's widely distributed in cells especially in mammalian liver. Carboxylesterase 5 (CES5), also known as cauxin or CES7, is one of CES enzyme families exerting role in catalyzing hydrolytic and transesterfication reactions with broad substrat specifity. CES5 is reported in high concentrations in the urine of adult male cats, and within a protein complex of mammalian male epididymal fluids. Roles for carboxylesterase 5 may include regulating urinary levels of cat pheromone, catalyzing lipid transfer reactions within mammalian male reproductive fluids, and protecting neural tissue from drugs and xenobiotics.