Human Acid sphingomyelinase Baculovirus-Insect Overexpression Lysate 产品信息
Product Description
This Human Acid sphingomyelinase overexpression lysate was created in Baculovirus-Insect Cells and intented for use as a Western blot (WB) positive control. Purification of Acid sphingomyelinase protein (Cat: 11087-H08B1) from the overexpression lysate was verified.
Expression Host
Baculovirus-Insect Cells
Sequence Information
A DNA sequence encoding the human SMPD1 (BAD93012.1) (Met1-Pro628) was expressed with a polyhistidine tag at the C-terminus.
Molecule Mass
The recombinant human SMPD1 consists of 593 amino acids and predicts a molecular mass of 66.3 kDa.
Human Acid sphingomyelinase Baculovirus-Insect Overexpression Lysate Usage Guide
Preparation Method
Cell lysate was prepared by homogenization of the over-expressed cells 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.
Lysis Buffer
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.
Recommend Usage
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.
Sample Buffer
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
Human Acid sphingomyelinase Baculovirus-Insect Overexpression Lysate Alternative Names
Human ASM Overexpression Lysate;Human ASMASE Overexpression Lysate;Human NPD Overexpression Lysate
Acid sphingomyelinase Background Information
Sphingomyelin phosphodiesterase 1 (SMPD1) , also known as ASM ( acid sphingomyelinase ), is a member of the acid sphingomyelinase family of enzymes. Three isoforms have been identified, isoform 1 is 631 amino acids (aa) in length as the pro form, while Isoform 2 and isoform 3 have lost catalytic activity. The active SMPD1 isoform 1 contains one saposin B-type domain that likely interacts with sphingomyelin, and a catalytic region. Human SMPD1 is 86% aa identical to mouse SMPD1. SMPD1 is a monomeric lysosomal enzyme that converts sphingomyelin (a plasma membrane lipid ) into ceramide through the removal of phosphorylcholine. This generates second messenger components that participate in signal transduction. Defects in SMPD1 are the cause of Niemann-Pick disease type A (NPA) and type B (NPB), also known as Niemann-Pick disease classical infantile form and Niemann-Pick disease visceral form. Niemann-Pick disease is a clinically and genetically heterogeneous recessive disorder. NPB has little if any neurologic involvement and patients may survive into adulthood.
Full Name
sphingomyelin phosphodiesterase 1
References
Schuchman E.H., et al.,(1991), Human acid sphingomyelinase. Isolation, nucleotide sequence and expression of the full-length and alternatively spliced cDNAs. J. Biol. Chem. 266:8531-8539. Newrzella D., et al., (1992), Molecular cloning of the acid sphingomyelinase of the mouse and the organization and complete nucleotide sequence of the gene.Biol. Chem. Hoppe-Seyler 373:1233-1238. Schuchman E.H., et al.,(1992), Structural organization and complete nucleotide sequence of the gene encoding human acid sphingomyelinase (SMPD1).Genomics 12:197-205.
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