This Human Von Willebrand Factor overexpression lysate was created in CHO Cells and intented for use as a Western blot (WB) positive control. Purification of Von Willebrand Factor protein (Cat: 10973-H08C) from the overexpression lysate was verified.
A DNA sequence encoding the pro form of human von Willebrand factor (NP_000543.2) (Met 1-Lys 2813) was expressed with a C-terminal polyhistidine tag.
The secreted recombinant human vWF consists of 2802 amino acids after removal of the signal peptide and has a predicted molecular mass of 308 kDa. Purified rhvWf exists as both the pro form with the pro peptide (307 kDa) and the mature form (226 kDa), which migrates as doublets with apparent molecular mass of 260 and 350 kDa respectively in SDS-PAGE under reducing conditions due to glycosylation.
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.
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.
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 F8VWF Overexpression Lysate;Human Von Willebrand Factor Overexpression Lysate;Human VWD Overexpression Lysate
Von Willebrand Factor (VWF) is a multimeric glycoprotein involved in hemostasis in blood, binds receptors on the surface of platelets and in connective tissue, thereby mediating the adhesion of platelets to sites of vascular injury. From studies it appears that VWF protein uncoils under these circumstances, decelerating passing platelets. VWF protein is deficient or defective in von Willebrand disease (VWD) and is involved in a large number of other diseases, including thrombosis, thrombotic thrombocytopenic purpura, Stroke, Heyde's syndrome, possibly hemolytic-uremic syndrome and so on.
Sadler JE. (1998) Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem, 67: 395-424.Batlle J, et al. (2009) Von Willebrand factor/factor VIII concentrates in the treatment of von Willebrand disease. Blood Coagul Fibrinolysis. 20(2):89-100.Sadler JE. (2009) von Willebrand factor assembly and secretion. J Thromb Haemost. 7 Suppl 1:24-7.Auton M, et al. (2010) The mechanism of VWF-mediated platelet GPIbalpha binding. Biophys J. 99(4):1192-201.Bowen DJ. (2010) Sugar targets VWF for the chop. Blood. 115(13):2565.Lpez JA, et al. (2010) VWF self-association: more bands for the buck. Blood. 116(19):3693-4.