This Mouse CD64 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD64 protein (Cat: 50086-M27H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain (Met 1-Pro 297) of mouse FCGR1 (NP_034316.1) was fused with a c-terminal polyhistidine tagged AVI tag at the C-terminus.
The recombinant mouse FCGR1 consists of 308 amino acids after removal of the signal peptide and has a predicted molecular mass of 34.3 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rm FCGR1 is approximately 45-50 kDa due to glycosylation.
Mouse CD64 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.
High affinity immunoglobulin gamma Fc receptor I, also known as FCGR1 and CD64, is an integral membraneglycoprotein and a member of the immunoglobulin superfamily. CD64 is a high affinity receptor for the Fc region of IgG gamma and functions in both innate and adaptive immune responses. Receptors that recognize the Fc portion of IgG function in the regulation of immune response and are divided into three classes designated CD64, CD32, and CD16. CD64 is structurally composed of asignal peptidethat allows its transport to the surface of a cell, threeextracellularimmunoglobulin domainsof the C2-type that it uses to bind antibody, a hydrophobictransmembrane domain, and a short cytoplasmic tail. CD64 isconstitutivelyfound on only macrophages and monocytes, but treatment of polymorphonuclear leukocyteswith cytokines likeIFNγandG-CSFcan induce CD64 expression on these cells. The inactivation of the mouse CD64 resulted in a wide range of defects in antibody Fc-dependent functions. Mouse CD64 is an early participant in Fc-dependent cell activation and in the development of immune responses.