|Baculovirus-Insect Cell lysate that Human AMPK (G1/B2/A2) Heterotrimer transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|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.|
|Met & Met & His|
|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 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.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt at -70℃|
|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. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.