This Human AMPK alpha 1 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of AMPK alpha 1 protein (Cat: CT006-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 PRKAA2 (P54646) (Met 1-Arg 552) 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 / PRKAA2) has a calculated molecular mass of 158 (38+30+90) KDa. The apparent molecular mass is approximately 35, 37 & 95 KDa respectively 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.
Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube.
2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min.
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.