This Mouse Semaphorin 4D overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Semaphorin 4D protein (Cat: 50642-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse SEMA4D (NP_038688.2) extracellular domain (Met 1-Arg 733) was expressed, with a C-terminal polyhistidine tag.
The secreted recombinant mouse SEMA4D comprises 721 amino acids and has a calculated molecular mass of 80.2 kDa. As a result of glycosylation, the recombinant protein migrates as an approximately 100-110 kDa band in SDS-PAGE under reducing conditions.
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.
Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. VEGF and SEMA4D had a positive correlation with the malignant degree of ovarian cancer, and SEMA4D can serve as an independent prognostic factor. SEMA4D was the first semaphorin described to have immune functions and serves important roles in T cell priming, antibody production, and cell-to-cell adhesion. Proteolytic cleavage of SEMA4D from the cell surface gives rise to a soluble fragment of SEMA4D (sSEMA4D). Similar to the transmembranal form, sSEMA4D is thought to have immunoregulatory properties.