Human CD14 HEK293 Overexpression Lysate: Product Information
This Human CD14 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD14 protein (Cat: 10073-H01H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain (Thr 20-Cys 352) of the mature form of human CD14 (NP_000582.1) was fused to the Fc region of human IgG1 at the N-terminus.
The recombinant human Fc/CD14 chimera is a disulfide-linked homodimeric protein. The reduced monomer consists of 570 amino acids and has a calculated molecular mass of 62.4 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rhFc/CD14 monomer is approximately 75-85 kDa due to the glycosylation.
Human CD14 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 CD14 HEK293 Overexpression Lysate: Synonyms
Human CD14 antigen Overexpression Lysate; Human monocyte differentiation antigen CD14 Overexpression Lysate
CD14 Background Information
The cluster of differentiation (CD) system is commonly used as cell markers in immunophenotyping. Different kinds of cells in the immune system can be identified through the surface CD molecules associating with the immune function of the cell. There are more than 320 CD unique clusters and subclusters have been identified. Some of the CD molecules serve as receptors or ligands important to the cell through initiating a signal cascade which then alter the behavior of the cell. Some CD proteins do not take part in cell signal process but have other functions such as cell adhesion. Cluster of differentiation 14 (CD14) is a member of the CD system. It takes its name from its inclusion in the CD molecule surface marker proteins. CD14 exists in two forms: a form anchored into the membrane or a soluble form. CD14 was found expressed in macrophages, neutrophil granulocyte and dendritic cells. The major function is to serve as a co-receptor (along with TLR4 and MD-2) for the bacterial lipopolysaccharide (LPS) and other pathogen-associated molecular patterns.
NF-kB (NFkB) Pathway
Zola H, et al. (2007) CD molecules 2006-human cell differentiation molecules. J Immunol Methods. 318 (1-2): 1-5.
Ho IC, et al. (2009) GATA3 and the T-cell lineage: essential functions before and after T-helper-2-cell differentiation. Nat Rev Immunol. 9 (2): 125-35.
Matesanz-Isabel J, et al. (2011) New B-cell CD molecules. Immunology Letters.134 (2): 104-12.
SD Wright, et al. (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 249 (4975): 1431-3.
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