This Human Protein C overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Protein C protein (Cat: 10196-H08H) from the overexpression lysate was verified.
A DNA sequence encoding the human PROC (P04070) (Met1-Pro461) was expressed with a C-terminal polyhistidine tag.
The recombinant human PROC comprises 454 amino acids and has a predicted molecular mass of 51.5 kDa. The apparent molecular mass of the protein is approximately 56-64 and 45 kDa 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 APC Overexpression Lysate;Human PC Overexpression Lysate;Human PROC Overexpression Lysate;Human PROC1 Overexpression Lysate;Human THPH3 Overexpression Lysate;Human THPH4 Overexpression Lysate