Human HGF Activator/HGFA HEK293 Overexpression Lysate

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Human HGF Activator/HGFA HEK293 Overexpression Lysate: Product Information

Product Description
This Human HGF Activator/HGFA overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of HGF Activator/HGFA protein (Cat: 10329-H08H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Species
Human
Sequence Information
A DNA sequence encoding the human HGF activator precursor (NP_001519.1) (Met 1-Ser 655) was expressed with a fused polyhistidine-tag at the C-terminus.
Molecule Mass
The recombinant human HGF activator consists of 631 amino acids and has a predicted molecular mass of 68.2 kDa. As a result of proteolysis or autocatalysis and glycosylation, it migrates as four bands (34, 37, 65, 105 kDa) in SDS-PAGE under reducing conditions, corresponding to the active long chain, uncleaved single chain (long chain + short chain), pro doamin and the full-length pro form of HGFA respectively.

Human HGF Activator/HGFA 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.

Human HGF Activator/HGFA HEK293 Overexpression Lysate: Alternative Names

Human HGFA Overexpression Lysate; Human MGC138395 Overexpression Lysate; Human MGC138397 Overexpression Lysate; Human RP11-529E10.2 Overexpression Lysate

HGF Activator/HGFA Background Information

HGF activator (HGFA) is a serum-derived serine protease and belongs to the peptidase family S1.HGFA is responsible for the conversion of hepatocyte growth factor (HGF), from the inactive single-chain precursor to the active heterodimeric form, which is a potent mitogen, motogen, and morphogen for liver cells, epithelial cells, and endothelial cells. HGFA is synthesized and secreted by the liver and circulates in the plasma as an inactive single-chain zymogen in normal states. The zymogen is cleaved by thrombin or thermolysin through the endoproteolytic process and forms an active heterodimer linked by a disulfide bond. In turn, the active protease can be inhibited by HGFA inhibitors (HAIs) including HAI-1 and HAI-2. In addition, the HGFA zymogen acquires a strong affinity upon activation and thus may ensure the local action in tissue regeneration in liver, kidney and skin. It has been reported that activation of HGF is a critical limiting step in the HGF/SF-induced signaling pathway mediated by Met, and accordingly, aberrant expression of HGFA is implicated in tumorigenesis and progression.
References

    1.  Shimomura, T. et al., 1993, J. Biol. Chem. 268: 22927-22932.

    2.  Miyazawa, K. et al., 1996, J. Biol. Chem. 271 : 3615-3618.

    3.  Shia, S. et al., 2005, J. Mol. Biol. 346: 1335-1349.

    4.  Kataoka, H. et al., 2000, J. Biol. Chem. 275: 40453-40462.

    5.  Tjin, E.P. et al., 2006, Blood. 107: 760-768.

    6.  Kitajima, Y. et al., 2000, Cancer. Res. 60: 6148-6159.

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