Product Description
This Human DR4 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of DR4 protein (Cat: 10408-H03H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the human TNFRSF10A (NP_003835.2) extracellular domain (Met 1-Asn 239) was fused with the C-terminal polyhistidine-tagged Fc region of human IgG1 at the C-terminus.
Molecule Mass
The recombinant human TNFRSF10A/Fc is a disulfide-linked homodimer. The reduced monomer consists of 378 amino acids and has a predicted molecular mass of 42 kDa. As a result of glycosylation, the apparent molecular mass of rh TNFRSF10A/Fc monomer migrates with an apparent molecular mass of 47 kDa in SDS-PAGE under reducing conditions.
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 APO2 Overexpression Lysate;Human CD261 Overexpression Lysate;Human DR4 Overexpression Lysate;Human MGC9365 Overexpression Lysate;Human TNFRSF10A Overexpression Lysate;Human TRAILR-1 Overexpression Lysate;Human TRAILR1 Overexpression Lysate
Tumor necrosis factor receptor superfamily, member 1a (TRAIL R1), also known as TRAIL receptor 1 (TRAIL R1) or CD261 antigen, is a member of the TNF-receptor superfamily. This receptor is activated by tumor necrosis factor-related apoptosis inducing ligand (TNFSF1/TRAIL), and thus transduces cell death signal and induces cell apoptosis. Studies with FADD-deficient mice suggested that FADD, a death domain containing adaptor protein, is required for the apoptosis mediated by this protein. TRAIL R1/CD261/TNFRSF1A serves as a receptor for the cytotoxic ligand TNFSF1/TRAIL. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death-inducing signaling complex (DISC) performs caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases) mediating apoptosis. TRAIL R1 can promote the activation of NF-kappa-B. TRAIL R1/CD261/TNFRSF1A induces apoptosis of many transformed cell lines but not of normal tissues, even though its death domain-containing receptor, DR4, is expressed on both cell types.
Immune Checkpoint Immunotherapy Cancer Immunotherapy Targeted Therapy References
Kimberley FC, et al. (2005) Following a TRAIL: update on a ligand and its five receptors.". Cell Res. 14 (5): 359–72. Schneider P, et al. (1997) Characterization of two receptors for TRAIL.". FEBS Lett. 416 (3): 329–34.