Human AMPK (G1/B2/A1) Heterotrimer Baculovirus-Insect cells Overexpression Lysate: 产品信息
This Human AMPK (G1/B2/A1) Heterotrimer overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of AMPK (G1/B2/A1) Heterotrimer protein (Cat: CT005-H0907B) from the overexpression lysate was verified.
A DNA sequence encoding the human PRKAG1 (P54619) (Met 1-Pro 331), constructed the plasmid 1; A DNA sequence encoding the human PRKAB2 (O43741) (Met 1-Ile 272), constructed the plasmid 2; A DNA sequence encoding the human PRKAA1 (Q13131-1) (Met 1-Gln 559) was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus, constructed the plasmid 3. The three plasmids were co-expressed and the heterotrimer was purified.
The recombinant heterotrimer of human AMPK (PRKAG1 / PRKAB2 / PRKAA1) has a calculated molecular mass of 160 (38+30+92) KDa. The apparent molecular mass is approximately 35, 37 & 95 KDa respectively in SDS-PAGE under reducing conditions.
Human AMPK (G1/B2/A1) Heterotrimer Baculovirus-Insect cells 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.