Human CD3 epsilon/CD3e HEK293 Overexpression Lysate: Product Information
This Human CD3 epsilon/CD3e overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD3 epsilon/CD3e protein (Cat: 10977-H03H) from the overexpression lysate was verified.
A DNA sequence encoding the human CD3E (Met 1-Asp126) was fused with the C-terminal polyhistidine-tagged Fc region of human IgG1 at the C-terminus.
The secreted recombinant human CD3E is a disulfide-linked homodimeric protein. The reduced monomer comprises 355 amino acids and has a predicted molecular mass of 40 kDa. As a result of glycosylation, it migrates as an approximately 44-53 and 38 kDa band in SDS-PAGE under reducing conditions.
Human CD3 epsilon/CD3e HEK293 Overexpression Lysate: Usage Guide
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).
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.
Western Blot (WB) Optimal dilutions/concentrations should be determined by the end user.
Human CD3 epsilon/CD3e HEK293 Overexpression Lysate: Alternative Names
Human CD3 epsilon Overexpression Lysate; Human IMD18 Overexpression Lysate; Human T3E Overexpression Lysate; Human TCRE Overexpression Lysate
CD3 epsilon/CD3e Background Information
T-cell surface glycoprotein CD3 epsilon chain, also known as CD3E, is a single-pass type I membrane protein. CD3E contains 1 Ig-like (immunoglobulin-like) domain and 1 ITAM domain. CD3E, together with CD3-gamma, CD3-delta and CD3-zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T cell receptor-CD3 complex. The CD3 epsilon subunit of the T cell receptor (TCR) complex contains two defined signaling domains, a proline-rich sequence and an immune tyrosine activation motifs (ITAMs), and this complex undergoes a conformational change upon ligand binding that is thought to be important for the activation of T cells. In the CD3 epsilon mutant mice, all stages of T cell development and activation that are TCR-dependent were impaired, but not eliminated, including activation of mature naïve T cells with the MHCII presented superantigen, staphylococcal enterotoxin B, or with a strong TCR cross-linking antibody specific for either TCR-Cbeta or CD3 epsilon. T cell receptor-CD3 complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. This complex is critical for T-cell development and function, and represents one of the most complex transmembrane receptors. CD3E plays an essential role in T-cell development, and defects in CD3E gene cause severe immunodeficiency. Homozygous mutations in CD3D and CD3E genes lead to a complete block in T-cell development and thus to an early-onset severe combined immunodeficiency phenotype.
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