This Rhesus CD3D & CD3E Heterodimer overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD3D & CD3E Heterodimer protein (Cat: CT032-C0323H) from the overexpression lysate was verified.
A DNA sequence encoding the rhesus CD3D extracellular domain (F6WI60)(Met1-Ala105) with the C-terminal flag-tagged Fc region of human IgG1 at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the rhesus CD3E extracellular domain(XP_014971302.1) (Met1-Asp117) was fused with the C-terminal his-tagged Fc region of human IgG1 at the C-terminus, constructed the plasmid 2. The two plasmids were co-expressed and the CD3D&CD3E heterodimer was purified.
The recombinant heterodimer of rhesus CD3D&CD3E comprises 679 (346+333) amino acids and has a calculated molecular mass of 76.7 (39.1+ 37.6) KDa. The apparent molecular mass of rhesus CD3D&CD3E heterodimer is approximately 52 & 43 KDa respectively in SDS-PAGE.
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.