Human Acid sphingomyelinase/SMPD1 Baculovirus-Insect cells Overexpression Lysate: Product Information
This Human Acid sphingomyelinase/SMPD1 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of Acid sphingomyelinase/SMPD1 protein (Cat: 11087-H08B) from the overexpression lysate was verified.
A DNA sequence encoding the full length of human SMPD1 isoform 1 (NP_000534.3) (Met 1-Cys 631) was expressed, fused with a polyhistidine tag at the C-terminus.
The secreted recombinant human SMPD1 consists of 518 amino acids and predicts a molecular mass of 65 kDa as estimated by SDS-PAGE under reducing conditions.
Human Acid sphingomyelinase/SMPD1 Baculovirus-Insect cells Overexpression Lysate: Usage Guide
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.
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.
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.
Western Blot (WB) Optimal dilutions/concentrations should be determined by the end user.
Human Acid sphingomyelinase/SMPD1 Baculovirus-Insect cells Overexpression Lysate: Alternative Names
Human ASM Overexpression Lysate; Human ASMASE Overexpression Lysate; Human NPD Overexpression Lysate
Acid sphingomyelinase/SMPD1 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.
sphingomyelin phosphodiesterase 1
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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