This Human IL-35 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of IL-35 protein (Cat: CT065-H2508H) from the overexpression lysate was verified.
A DNA sequence encoding the human IL27B (NP_005746.2)(Met1-Lys229) was fused with a flag tag at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the human IL12A (NP_000873.2)(Met1-Ser219) was fused with a polyhistidine tag at the C-terminus, constructed the plasmid 2. The two plasmids were co-expressed and the human IL27B&IL12A heterodimer was purified.
The recombinant heterodimer of human IL27B&IL12A comprises 425 (217+208) amino acids and has a calculated molecular mass of 48.3(24.3+24) KDa.
Human IL-35 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.
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).
稳定性 & 储存条件
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 IL-35 HEK293 Overexpression Lysate: 别称
Human CLMF Overexpression Lysate; Human IL-12A Overexpression Lysate; Human IL-35 Overexpression Lysate; Human Interleukin-35 Overexpression Lysate; Human NFSK Overexpression Lysate; Human NKSF1 Overexpression Lysate; Human P35 Overexpression Lysate
IL12A is a subunit of a cytokine that acts on T and natural killer cells and has a broad array of biological activities. The cytokine is a disulfide-linked heterodimer composed of the 35-kD subunit encoded by this gene, and a 40-kD subunit that is a member of the cytokine receptor family. IL12A, together with IL27B, form a disulfide-linked heterodimer: IL12A&IL27B. IL12A&IL27B is required for the T-cell-independent induction of IFN-gamma and is important for the differentiation of both Th1 and Th2 cells. The responses of lymphocytes to this cytokine are mediated by the activator of transcription protein STAT4. Nitric oxide synthase 2A (NOS2A/NOS2) is found to be required for the signaling process of IL12A&IL27B in innate immunity.
IL12 signaling pathway
Wolf S.F., et al.,(1991), Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. J. Immunol. 146:3074-3081.
Gubler U., et al., (1991), Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.Proc. Natl. Acad. Sci. U.S.A. 88:4143-4147.
Batten M., et al.,(2007), The biology and therapeutic potential of interleukin 27.J. Mol. Med. 85:661-672.