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Human IL-35 (IL12A & IL27B) HEK293 Cell Lysate (WB positive control)

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Human IL35 Transfected / Overexpression Cell Lysate Product Information
Expressed Host:Human Cells
Product Description:Human Cell lysate that Human IL35 / Interleukin-35 transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).
Sequence information:A DNA sequence encoding the human IL35 complex composed of IL27B subunit (NP_005746.2) (Met 1-Lys 229) and the mature form of human IL12 p35 subunit (P29459) (Arg 23-Ser 219) linked by a polypeptide linker was fused with the Fc region of human IgG1 at the C-terminus.
Predicted N Terminal:Arg 21
Molecule Mass:The recombinant human IL35/Fc is a disulfide-linked homodimeric protein. The reduced monomer consists of 645 amino acids and has a predicted molecular mass of 73.4 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rhIL35/Fc monomer is approximately 90-100 kDa due to glycosylation.
Human IL35 Transfected / Overexpression Cell Lysate Usage Guide
Preparation Method:Cell lysate was prepared by homogenization 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.
Lysis Buffer: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.
Quality Control Testing:12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.
Stability:Samples are stable for up to twelve months from date of receipt.
Recommend Usage: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. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.
Storage Buffer:1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Storage Instruction:Store at 4℃. After re-dissolution, aliquot and store at -80℃.
Application notes:Western blot (WB): Use at an assay dependent dilution.
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
IL-35 Background

The novel Ebi3-IL-12alpha heterodimeric cytokine has been designated interleukin-35 (IL-35), is a member IL12 family cytokine produced by regulatory T cells (Treg), but not by resting or activated effector T cells (Teff). IL-35 is a heterodimeric protein composed of IL-12α (P35) and IL-27β chains, which are encoded by two separate genes called IL12A and EBI3 (Epstein-Barr-virus-induced gene 3) respectively. Ectopic expression of IL-35 confers regulatory activity on naive T cells, whereas recombinant IL-35 suppresses T-cell proliferation. It identify IL-35 as a novel inhibitory cytokine that may be specifically produced by T(reg) cells and is required for maximal suppressive activity. IL-35 has biological activity and able to expand CD4+CD25+ Treg cells, suppress the proliferation of CD4+CD25- effector cells and inhibit Th17 cell polarization. IL-35 has been shown to be constitutively expressed by regulatory T (Treg) cells CD4(+)CD25(+)Foxp3(+) and suggested to contribute to their suppressive activity. IL-35 is a crucial mediator which provokes CD4+CD25+ T cell proliferation and IL-10 generation, another well-known anti-inflammatory cytokine, along with TGFbeta cytokine. IL-35 is a cytokine can downregulate Th17 cell development and inhibit autoimmune inflammation. It inhibited the differentiation of Th17 cells in vitro. In vivo, IL-35 effectively attenuated established collagen-induced arthritis in mice, with concomitant suppression of IL-17 production but enhanced IFN-gamma synthesis. Thus, IL-35 is a novel anti-inflammatory cytokine suppressing the immune response through the expansion of regulatory T cells and suppression of Th17 cell development.

Human IL-35 References
  • Niedbala W, et al. (2007) IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells. Eur J Immunol. 37(11): 3021-9.
  • Collison LW, et al. (2007) The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature. 450(7169): 566-9.
  • Castellani ML, et al. (2010) IL-35, an anti-inflammatory cytokine which expands CD4+CD25+ Treg Cells. J Biol Regul Homeost Agents. 24(2): 131-5.
  • Kochetkova I, et al. (2010) IL-35 stimulation of CD39+ regulatory T cells confers protection against collagen II-induced arthritis via the production of IL-10. J Immunol. 184(12): 7144-53.
  • Seyerl M, et al. (2010) Human rhinoviruses induce IL-35-producing Treg via induction of B7-H1 (CD274) and sialoadhesin (CD169) on DC. Eur J Immunol. 40(2): 321-9.
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    Catalog: 10705-H02HL-300
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