This Mouse NK1.1/KLRB1F overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of NK1.1/KLRB1F protein (Cat: 50815-M01H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of mouse KLRB1F (Q8VD98) (Gln 67-Val 217) was fused with the Fc region of human IgG1 at the N-terminus.
The recombinant mouse KLRB1F/Fc is a disulfide-linked homodimer. The reduced monomer comprises 411 amino acids and has a calculated molecular mass of 46 kDa. As a result of glycosylation, the apparent molecular mass of the monomer is approximately 52 kDa 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.