This Mouse PDK4 overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of PDK4 protein (Cat: 51021-M20B) from the overexpression lysate was verified.
A DNA sequence encoding the mouse PDK4 (O70571) (Met1-Val412) was expressed with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
The recombinant mouse PDK4/GST chimera consists of 649 amino acids and has a calculated molecular mass of 74.4 kDa. The recombinant protein migrates as an approximately 65 kDa band 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.
Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates the TCA cycle.PDK4, a vital mitochondrial protein, controls the switch between glycolysis and oxidative phosphorylation based upon nutrient availability.Pyruvate dehydrogenase kinase 4 (PDK4) mRNA has been reported as an up-regulated gene in the heart and skeletal muscle of carnitine-deficient juvenile visceral steatosis (JVS) mice under fed conditions. PDK4 plays an important role in the inhibition of glucose oxidation via the phosphorylation of pyruvate dehydrogenase complex (PDC).PDK4 gene expression is stimulated by thyroid hormone (T(3)), glucocorticoids, and long chain fatty acids.