This Rat IL-23 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of IL-23 protein (Cat: CT045-R08H) from the overexpression lysate was verified.
A DNA sequence encoding the rat IL23A (NP_569094.1) (Met1-Ala196) was fused with a polyhistidine tag at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the rat IL12B (EDM04146.1) (Met1-Ser335) was expressed with a polyhistidine tag at the C-terminus, constructed the plasmid 2. The two plasmids were co-expressed and the rat IL23A & the rat IL12B heterodimer was purified.
The recombinant rat IL23A & the rat IL12B comprises 510 (186+324) amino acids and has a calculated molecular mass of 58.2 (21+37.2) KDa. The apparent molecular mass of the rat IL23A & the rat IL12B heterodimer is approximately 47.08, 43.29, 27.67 and 24.75 KDa respectively in SDS-PAGE.
Rat IL-23 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.