Product Description
This Human CD25 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD25 protein (Cat: 10165-H02H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the amino acid residues (Met 1-Cys 213) of the human IL2 receptor α chain (NP_000408.1) precursor was expressed with C-terminal fused human IgG1 Fc region.
Molecule Mass
The mature recombinant human IL2Rα/Fc is a disulfide-linked homodimeric protein generated by proteolytic removal of the signal peptide. The reduced monomer consists of 430 amino acids and has a predicted molecular mass of 48.5 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rhIL2Rα/Fc monomer is approximately 72 kDa due to glycosylation.
Preparation Method
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.
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.
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.
Sample Buffer
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.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
Human CD25 Overexpression Lysate;Human IDDM10 Overexpression Lysate;Human IL-2RA Overexpression Lysate;Human IL2R Overexpression Lysate;Human p55 Overexpression Lysate;Human TCGFR Overexpression Lysate
CD25 (alpha-chain of IL-2 receptor, or IL2RA), is a type I transmembrane glycoprotein with a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic domain. IL2RA is expressed on activated T cells and regulatory T cells, and is capable of binding IL2 with low affinity by itself. However, a ligand-induced high affinity heterotrimeric receptor complex is produced when IL2RA is associated non-covelently with the IL2 receptor beta and gamma chain, and subsequently initiates the intacellular signal pathways such as MAPK or JAK/STAT. On dendritic cells (DC), CD25 has been previously regarded as an activation marker, while both murine and human DC can express CD25, they do not express the beta-chain of the IL-2 receptor, which is indispensable for the execution of IL-2 signaling. The IL2RA (CD25) gene is a substantial component of the high-affinity receptor molecule highly expressed by activated T lymphocytes. Recently, a strong evidence was obtained for the involvement of IL-2RA in conferring susceptibility to type 1 diabetes (T1D). Cancer growth and development is associated with the stimulation of the innate immune system, including enhanced interleukin 2 receptor (IL-2R) expression in immune cells and its shedding into the circulation in a soluble form of sIL-2Ralpha. In most haematological malignancies, including different types of leukaemias and lymphomas, sIL-2Ralpha has been found to be released directly from the surface of neoplastic cells thus reflecting the tumour bulk, turnover and activity. Several studies have proved that not only lymphoid cancer cells, but also some non-lymphoid cancer cells, express IL-2R on their surface. They include malignant melanoma and carcinomas of the kidney, head and neck, oesophagus and lung. Thus, sIL-2Ralpha is elevated in most proliferative disturbances of the hematopoietic system and in many solid tumors.
Immune Checkpoint Immunotherapy Cancer Immunotherapy Targeted Therapy References
Driesen J, et al. (2008) CD25 as an immune regulatory molecule expressed on myeloid dendritic cells. Immunobiology. 213(9-10): 849-58. Olejniczak K, et al. (2008) Biological properties of interleukin 2 and its role in pathogenesis of selected diseases--a review. Med Sci Monit. 14(10): RA179-89. Chistiakov DA, et al. (2008) The crucial role of IL-2/IL-2RA-mediated immune regulation in the pathogenesis of type 1 diabetes, an evidence coming from genetic and animal model studies. Immunol Lett. 118(1): 1-5. Bien E, et al. (2008) Serum soluble interleukin 2 receptor alpha in human cancer of adults and children: a review. Biomarkers. 13(1): 1-26. 
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