This Blomia tropicalis Blot21 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Blot21 protein (Cat: 69065-A08H) from the overexpression lysate was verified.
A DNA sequence encoding the Blot21 (AAX34047.1) (Met1-Glu129) was expressed with a polyhistidine tag at the C-terminus.
The recombinant Blot21 consists of 140 amino acids and predicts a molecular mass of 16.3 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.