This Mouse FCGRT & B2M Heterodimer overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of FCGRT & B2M Heterodimer protein (Cat: CT029-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain (Met 1-Ser 297) of mouse FCGRT (Q61559) was fused with a polyhistidine tag at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the mouse B2M (P01887,105Ala/Asp) (Met 1-Met 119) constructed the plasmid 2. The two plasmids were co-expressed and the FCGRT / B2M heterodimer was purified
The recombinant heterodimer of mouse FCGRT / B2M comprises 385 ( 285 + 100 ) amino acids and has a calculated molecular mass of 43.5 ( 31.8 + 11.7 ) kDa. The apparent molecular mass of FCGRT / B2M heterodimer is approximately 35 & 12 kDa respectively in SDS-PAGE under reducing conditions
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.