This Rhesus IL-12 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of IL-12 protein (Cat: CT045-C08H) from the overexpression lysate was verified.
A DNA sequence encoding the rhesus IL12A (NP_001038199.1) (Met1-Ser219) was fused with a polyhistidine tag at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the rhesus IL12B (NP_001038190.1) (Met1-Ser328) was expressed with a polyhistidine tag at the C-terminus, constructed the plasmid 2. The two plasmids were co-expressed and the rhesus IL12A & the rhesus IL12B heterodimer was purified.
The recombinant rhesus IL12A & the rhesus IL12B comprises 525(208+317) amino acids and has a calculated molecular mass of 60.2(24+36.2) KDa.
Rhesus IL-12 HEK293 Overexpression Lysate: 使用指南
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.