Mouse ASAH2 HEK293 Overexpression Lysate 产品信息
Product Description
This Mouse ASAH2 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of ASAH2 protein (Cat: 50006-M07H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the Lumenal domain (Thr 34-Thr 756) of mouse ASAH2 (NP_061300.1) precursor was expressed with a N-terminal polyhistidine tag.
Molecule Mass
The secreted recombinant mouse ASAH2 consists of 739 amino acids and has a calculated molecular mass of 82 kDa. As a result of glycosylation, the recombinant protein migrates as an approximately 105-115 kDa protein in SDS-PAGE under reducing conditions.
Mouse ASAH2 HEK293 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.
Mouse ASAH2 HEK293 Overexpression Lysate Alternative Names
Mouse AI585898 Overexpression Lysate
ASAH2 Background Information
ASAH2 (N-acylsphingosine amidohydrolase 2), also known as neutral ceramidase, is a type II integral membrane protein that can be cleaved to produce a soluble secreted protein. The enzyme is abundant in the brush border membranes of the intestine, and also expressed in several tissues such as kidney, brain and liver. The primary structure of ASAH2/neutral ceramidase is highly conserved from bacteria to humans, however, there is a clear difference in the molecular architecture. The murine ASAH2 possesses ‘amucin box’, a Ser/Thr/Pro-rich domain glycosylated with O-glycans which is necessary to retain the enzyme on the plasma membrane as a type II integral protein. The major physiological function of ASAH2/neutral ceramidase is the metabolism of dietary sphingolipids, and thus plays a role in the generation of messenger molecules such as sphingosine and sphingosine 1-phosphate.
Full Name
N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2
References
Tani M, et al. (2000) Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase. A novel but highly conserved gene family of neutral/alkaline ceramidases. J Biol Chem. 275(15): 11229-34. Franzen R, et al. (2002) Nitric oxide induces neutral ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures. FEBS Lett. 532(3): 441-4. Kono M, et al. (2006) Neutral ceramidase encoded by the Asah2 gene is essential for the intestinal degradation of sphingolipids. J Biol Chem. 281(11): 7324-31.