Sphingolipids are hydrolyzed by ceramidases to yield sphingosine and fatty acids. These ceramidases are classified according to the pH range that supports their optimal activity. ASAH2 is a neutral ceramidase and key regulator of sphingolipid signaling metabolites at the cell surface, catalyzing the hydrolysis of the N-acyl linkage of ceramide at an optimal pH of 6.5-8.5. ASAH2 is a type II integral membrane protein that can be cleaved to yield a soluble secreted protein and acts as a repressor of apoptosis both by reducing C16-ceramide, thereby preventing ceramide-induced apoptosis, and generating sphingosine. Sphingosine exerts both mitogenic and apoptosis-inducing activities, and its phosphorylated form functions as an intra- and intercellular second messenger. ASAH2 is ubiquitously expressed primarily expressed with higher levels in the intestine, kidney, skeletal muscle and heart. Recent studies indicate that ASAH2 encoded neutral ceramidase is a key enzyme for the catabolism of dietary sphingolipids and regulates the levels of bioactive sphingolipid metabolites in the intestinal tract.
This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.
Name: Anti-ASAH2 antibody (NBP1-76360) Catalog #: Anti-ASAH2 antibody (NBP1-76360) Lot Number: Anti-ASAH2 antibody (NBP1-76360, Lot # 4743-0803) PO/Order Number: Click here to enter text.. WB Image Description (Please provide labels for all lanes): lane 1: human primary hepatocytes; lane 2: Hep G2 Sample Information: Cell Line or Tissue: human primary hepatocytes; and Hep G2 Species: human Treatment: No treatment Lysate Preparation: Date of lysate preparation: December 4, 2017 Lysis buffer used: 1X lysis buffer from Cell Signaling by adding PMSF Reducing agent: beta-mercaptoethanol, DTT If boiled (temperature/time): Yes Controls: Positive Control: No Negative Control: No Loading Control (please attach additional images if applicable): No Protein Amount Loaded per lane: 20 ug Antibody Storage Conditions: -20℃ Electrophoresis: Gel Percentage: 10% Electrophoresis Conditions: Tris-Glycine-SDS at room temperature Voltage: 120V Time: 2 hours Membrane Transfer: Method (Submersion/Semi-dry): wet transfer Membrane Type (PVDF/Nitrocellulose): Nitrocellulose Time: 2 hours Voltage: 100V Blocking: Blocking Solution: 5% milk in 1X TBST Time: 1 hour at room temperature Primary Antibody: Dilution: 1/1000 Diluent Buffer: 2.5% BSA Incubation Time: overnight Incubation Temperature: 4℃ Washing Conditions: Wash Solution: 1X TBST Time and Repetitions: 5 min each for 3 times Secondary Antibody Manufacturer and Catalog #: Promega, W401B, Lot # 0000187662 Secondary description: goat anti-rabbit secondary antibody Dilution: 1/2000 Diluent Buffer: 3% milk Incubation Time: 1 hour Incubation Temperature: room temperature
Detection Method: Detection: ECL (GE, cat # RPN2209, lot # 9838243) Procedure: Add equal volume of A and B, mix and apply on the membranes for 3-5 min before exposure Development Time: 45 seconds Molecular weight of band(s): ~ 70 kDa Experimental Concerns and Observations: A specific band around 70 kDa was observed
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