Mouse VCAM1 HEK293 Overexpression Lysate: Product Information
This Mouse VCAM1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of VCAM1 protein (Cat: 50163-M03H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain (Met 1-Glu 698) of mouse VCAM1 (NP_035823.3) precursor was fused with the C-terminal polyhistidine-tagged Fc region of human IgG1 at the C-terminus.
The recombinant mouse VCAM1/Fc is a disulfide-linked homodimer after removal of the signal peptide. The reduced monomer consists of 922 amino acids and has a predicted molecular mass of 102 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rmVCAM1/Fc monomer is approximately 110-120 kDa due to glycosylation.
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.
Vascular cell adhesion molecule 1 (VCAM-1), also known as CD16, is a cell surface sialoglycoprotein belonging to the immunoglobulin superfamily. Two forms of VCAM-1 with either six or seven extracellular Ig-like domains are generated by alternative splicing, with the longer form predominant. VCAM-1 is an endothelial ligand for very late antigen-4 (VLA-4) and α4ß7 integrin expressed on leukocytes, and thus mediates leukocyte-endothelial cell adhesion and signal transduction. VCAM-1 expression is induced on endothelial cells during inflammatory bowel disease, atherosclerosis, allograft rejection, infection, and asthmatic responses. During these responses, VCAM-1 forms a scaffold for leukocyte migration. VCAM-1 also activates signals within endothelial cells resulting in the opening of an "endothelial cell gate" through which leukocytes migrate. VCAM-1 has been identified as a potential anti-inflammatory therapeutic target, the hypothesis being that reduced expression of VCAM-1 will slow the development of atherosclerosis. In addition, VCAM-1-activated signals in endothelial cells are regulated by cytokines indicating that it is important to consider both endothelial cell adhesion molecule expression and function during inflammatory processes.
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