This Rhesus CD1C & B2M Heterodimer overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD1C & B2M Heterodimer protein (Cat: CT057-C08H) from the overexpression lysate was verified.
A DNA sequence encoding the rhesus CD1C(NP_001139005.1) (Met1-Phe298) of was expressed with a polyhistidine tag at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the rhesus B2M (NP_001040602.1) (Met1-Met119) constructed the plasmid 2. The two plasmids were co-expressed and the CD1C/B2M heterodimer was purified.
The recombinant heterodimer of rhesus CD1C&B2M comprises 388 (289+99) amino acids and has a calculated molecular mass of 44.7 (33.1+ 11.6) KDa.
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.