This Human HVEM overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of HVEM protein (Cat: 10334-H03H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain (Met 1-Val 202) of human HVEM (NP_003811.2) precursor was fused with the C-terminal polyhistidine-tagged Fc region of human IgG1 at the C-terminus.
The recombinant human HVEM/Fc is a disulfide-linked homodimeric protein after removal of the signal peptide. The reduced monomer consists of 413 amino acids and predicts a molecular mass of 45.4 kDa. By SDS-PAGE under reducing conditions, the apparent molecular mass of rh HVEM/Fc monomer is approximately 60-65 kDa due to glycosylation.
Human HVEM 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 HVEM HEK293 Overexpression Lysate: 别称
Human ATAR Overexpression Lysate; Human CD270 Overexpression Lysate; Human HVEA Overexpression Lysate; Human HVEM Overexpression Lysate; Human LIGHTR Overexpression Lysate; Human TR2 Overexpression Lysate
Herpesvirus entry mediator (HVEM), also referred to as TNFRSF14, TR2 (TNF receptor-like molecule) and ATAR (another TRAF-associated receptor), is a member of type I transmembrane protein belonging to the TNF-receptor superfamily. It is expressed on many immune cells, including T and B cells, NK cells, monocytes, and neutrophils. Two TNF superfamily ligands lymphotoxin α (TNF-β) and LIGHT (TNFSF14) are identified as cellular ligands for HVEM and initiate the positive signaling. However, recent studies have revealed that HVEM is also involved in the unique inhibitory signaling pathway for T cells through activating tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in B and T lymphocyte attenuator (BTLA). HVEM provides a stimulatory signal following engagement with LIGHT (TNFSF14) on T cells. In contrast, it can also provide an inhibitory signal to T cells when it binds the B and T lymphocyte attenuator (BTLA), a ligand member of the Immunoglobulin (Ig) superfamily. Thus, HVEM may be viewed as a molecular switch, capable of facilitating both stimulatory and inhibitory cosignaling in T cells. Substantial evidence from both human disease and from experimental mouse models has indicated that dysregulation of the LIGHT-HVEM-BTLA cosignaling pathway can cause inflammation in the lung and in mucosal tissues.