This Human IgG4 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of IgG4 protein (Cat: 13505-HNAH) from the overexpression lysate was verified.
A DNA sequence encoding the human IgG4 Fc region (P01861) (Glu 99-Lys 327, with mutation Ser 108 Pro) was expressed and purified.
The recombinant human IgG4-Fc consists of 229 amino acids and has a predicted molecular mass of 25.5 kDa. As a result of glycosylation, the apparent molecular mass of IgG4-Fc is approximately 35 kDa in SDS-PAGE under reducing conditions.
Human IgG4 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.