This Cynomolgus CD32A overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD32A protein (Cat: 90015-C27H) from the overexpression lysate was verified.
A DNA sequence encoding the Cynomolgus (Macaca fascicularis) CD32a (AAL92096.1) extracellular domain (Met 1-Gly 220) was fused with a c-terminal polyhistidine tagged AVI tag at the C-terminus.
The recombinant cynomolgus CD32a consists of 206 amino acids and has a calculated molecular mass of 22.9 kDa. The apparent molecular mass of the recombinant protein is approximately 33-35 kDa in SDS-PAGE under reducing conditions due to glycosylation.
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.