Human Urokinase/uPA HEK293 Overexpression Lysate: Product Information
This Human Urokinase/uPA overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Urokinase/uPA protein (Cat: 10815-H08H) from the overexpression lysate was verified.
A DNA sequence encoding the human PLAU (NP_002649.1) (Met 1-Leu 431) with a C-terminal polyhistidine tag was expressed.
The secreted recombinant human PLAU comprises 422 amino acids with a predicted molecular mass of 46 kDa. As a result of glycosylation, rhPLAU migrates as three bands corresponding to the long A chain, B chain and unprocessed full-length PLAU with the molecular mass of 18, 32 and 50 kDa respectively in SDS-PAGE under reducing conditions.
Human Urokinase/uPA HEK293 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 Urokinase/uPA HEK293 Overexpression Lysate: Alternative Names
Human ATF Overexpression Lysate; Human BDPLT5 Overexpression Lysate; Human QPD Overexpression Lysate; Human u-PA Overexpression Lysate; Human UPA Overexpression Lysate; Human URK Overexpression Lysate
Urokinase/uPA Background Information
Plasminogen activator, urokinase, also known as PLAU and uPA, is a serine protease which converts plasminogen to plasmin, a broad-spectrum protease active on extracellular matrix (ECM) components. It is involved in complement activation, cell migration, wound healing, and generation of localized extracellular proteolysis during tissue remodelling, pro-hormone conversion, carcinogenesis and neoplasia. Like many components of the blood coagulation, fibrinolytic and complement cascades, uPA has a modular structure, including three conserved domains: a growth factor-like domain (GFD, residues 1-49), a kringle domain (residues 5-131), linked by an interdomain linker or "connecting peptide" (CP, residues 132-158) to the serine protease domain (residues 159-411). uPA and its receptor (uPAR) have been implicated in a broad spectrum of pathophysiological processes, including fibrinolysis, proteolysis, inflammation, atherogenesis and plaque destabilization, all of which are involved in the pathogenesis of MI (myocardial infarction). The role of uPA is not only linked to its action as an enzyme. In fact, the mere binding of uPA on the cell surface also brings about two events that broaden the spectrum of its biological functions: (1) a conformational change of the receptor, which, in turn, affects its interaction with other proteins; (2) a signal transduction which modulates the expression of apoptosis-related genes. Besides its applications as a thrombolytic agent and as a prognostic marker for tumors, uPA may provide the basis for other therapies, as the structure of the receptor-binding domain of uPA has become a model for the design of anti-cancer molecules. Because of the causal involvment of uPA in cancer invasion and metastasis, the blockade of uPA interactions and activity with specific inhibitors is of interest for novel strategies in cancer therapy.
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