This Mouse B7-1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of B7-1 protein (Cat: 50446-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse CD80 (Q00609-1) extracellular domain (Met 1-Lys 245) was expressed, with a polyhistidine tag at the C-terminus.
The recombinant mouse CD80 consists of 219 amino acids and has a predicted molecular mass of 25.1 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rm CD80 is approximately 48-50 kDa due to glycosylation.
Mouse B7-1 HEK293 Overexpression Lysate: 使用指南
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.
The B-lymphocyte activation antigen B7-1 (referred to as B7), also known as CD8, is a member of cell surface immunoglobulin superfamily and is expressed on the surface of antigen-presenting cells including activated B cells, macrophages and dendritic cells. As costimulatory ligands, B7-1 which exists predominantly as dimer and the related protein B7-2, interact with the costimulatory receptors CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on T cells, and thus constitute one of the dominant pathways that regulate T cell activation and tolerance, cytokine production, and the generation of CTL. The B7/CD28/CTLA4 pathway has the ability to both positively and negatively regulate immune responses. CD8 is thus regarded as promising therapeutic targets for autoimmune diseases and various carcinomas.