Human CD3D & CD3E Heterodimer HEK293 Overexpression Lysate: 产品信息
This Human 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: CT038-H2508H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain (Met1-Ala105) of human CD3D (P04234-1) was fused with a flag tag at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the extracellular domain (Met1-Asp126) of human CD3E (P07766-1) was fused with a polyhistidine tag at the C-terminus, constructed the plasmid 2. The two plasmids were co-expressed and the human CD3D/CD3E heterodimer was purified.
The recombinant heterodimer of human CD3D&CD3E comprises 271 (126+145) amino acids and has a calculated molecular mass of 31.05 (14.4+16.65)KDa. As a result of glycosylation, the apparent molecular mass of human CD3D&CD3E heterodimer is approximately 20, 22 and 26 KDa in SDS-PAGE under reducing conditions.
Human CD3D & CD3E Heterodimer 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.