Purification and Quality Control The His-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns. The purified PPAR-alpha-LBD is greater than 95% homogeneous based on SDS-PAGE analysis.
Unit Definition (Activity) 1 unit equals 1 nanogram of purified protein. 20-100 units are sufficient for a ligand binding assay and 100 units are sufficient for a protein-protein interaction assay.
Applications PPAR-alpha has been applied in DNA and protein-protein interactions assays.
Formulation and Storage The protein is in 20mM Tris-HCl pH7.9,100mM NaCl, 0.2mM EDTA, 1mM DTT and 20% glycerol. Stored at -70°C before use. Avoid repeated freeze thaw cycles.
Background There is evidence that a group of closely related nuclear receptors, called peroxisome proliferator-activated receptors (PPARs), may be involved in chronic diseases such as diabetes, obesity, artherosclerosis and cancer. The PPARs were first cloned as the nuclear receptors that mediate the effects of synthetic compounds called peroxisome proliferators on gene transcription. It soon became clear that eicosanoids and fatty acids can also regulate gene transcription through PPARs. They bind a specific element in the promoter region of target genes only as a heterodimer with the receptor for 9- cis retinoic acid, RXR (retinoid X receptor). Binding of the ligand of either receptor can activate the complex, but binding of both ligands simultaneously is more potent (1). Three PPAR isotypes have been identified: α, β (δ) (also called NUC1) and γ. PPARα is expressed most in brown adipose tissue and liver, then kidney, heart and skeletal muscle. PPARγ is mainly expressed in adipose tissue, and to a lesser extent in colon, the immune system and the retina. PPARβ is found in many tissues but the highest expression is in the gut, kidney and heart (2). The target genes of PPARα are a relatively homogenous group of genes that participate in aspects of lipid catabolism such as fatty acid uptake through membranes, fatty acid binding in cells, fatty acid oxidation (in microsomes, peroxisomes and mitochondria) and lipoprotein assembly and transport (3).
