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: CT008-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 PRKAB1 (Q9Y478) (Met 1-Ile 270), 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 / PRKAB1 / PRKAA2) has a calculated molecular mass of 158 (38+30+90) KDa. The apparent molecular mass is approximately 40, 42 & 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.
The PRKAA1 gene encodes the catalytic alpha-subunit of 5' AMP-activated protein kinase (AMPK). AMPK is a cellular energy sensor that maintains energy homeostasis within the cell and is activated when the AMP/ATP ratio increases. Moreover, AMPK has been shown to be involved in inhibiting tumour growth and metastasis, and has also been implicated in the pathology of neurodegenerative and cardiac disorders. All 5 tested tag SNPs of the PRKAA1 gene (rs1336177, rs154268, rs385486, rs688293, and rs174991) were significantly associated with gastric cancer.An inhibitor of PRKAA1 promoted the secretion of IL6, whereas an agonist of PRKAA1 suppressed the production of IL6.