This Mouse Thy1 / CD90 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Thy1 / CD90 protein (Cat: 50461-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of mouse THY1 (NP_033408.1) without the propeptide (Met 1-Cys 131) was expressed, with a polyhistidine tag at the C-terminus.
The recombinant mouse THY1 consists of 123 amino acids and has a predicted molecular mass of 14.2 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rm THY1 is approximately 20-27 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.
Thy-1 membrane glycoprotein, also known as Thy-1 antigen, CD9 and THY1, is a cell membrane protein which contains 1 Ig-like V-type (immunoglobulin-like) domain. It is a glycophosphatidylinositol-linked glycoprotein expressed on the surface of neurons, thymocytes, subsets of fibroblasts, endothelial cells, mesangial cells and some hematopoietic cells. It has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells. Thy-1 is evolutionarily conserved, developmentally regulated, and often has dramatic effects on cell phenotype. Thy-1 is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored protein involved in T cell activation, neurite outgrowth, apoptosis, tumor suppression, wound healing, and fibrosis. To mediate these diverse effects, Thy-1 participates in multiple signaling cascades. Thy-1 is an important regulator of cell-cell and cell-matrix interactions, with important roles in nerve regeneration, metastasis, inflammation, and fibrosis.