Each antibody in the AMPK, ACC Antibody Sampler Kit, BioAssay™ recognizes only its specific target. The antibodies do not cross-react with other AMPK family members.

Background: AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in the regulation of energy homeostasis (1). AMPK is a heterotrimeric complex composed of a catalytic subunit and regulatory and subunits, each of which is encoded by two or three distinct genes ( 1, 2; 1, 2; 1, 2, 3)(2). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia and ischemia (1). The tumor suppressor LKB1, in association with accessory proteins STRAD and MO25, phosphorylates AMPK at Thr172 in the activation loop and this phosphorylation is required for AMPK activation (3-5). AMPK is also phosphorylated at Thr258 and Ser485 (for 1; Ser491 for 2). The upstream kinase and biological significance of these phosphorylation events have yet to be elucidated (6). The 1 subunit is post- translationally modified by myristoylation and multi-site phosphorylation including Ser24/25, Ser96, Ser101 and Ser182 (6,7). Phosphorylation at Ser108 of the 1 subunit seems to be required for the activation of AMPK enzyme, while phosphorylation ot Ser24/25 and Ser182 affects AMPK localization (7). Accumulating evidence indicates that AMPK not only regulates the metabolism of fatty acids and glycogen, but also modulates protein synthesis and cell growth through EF2 and TSC2/mTOR pathways, as well as blood flow via eNOS/nNOS (1). Acetyl-CoA carboxylase (ACC) catalyzes the pivotal step of the fatty acid synthesis pathway. The 265kD ACC is the predominant isoform found in liver, adipocytes and mammary gland, while the 280kD ACC is the major isoform in skeletal muscle and heart (8). Phosphorylation by AMPK at Ser79 or by PKA at Ser1200 inhibits the enzymatic activity of ACC (9). ACC is a potential target of anti-obesity drugs (10,11).

Polyclonal antibodies are produced by immunizing rabbits with synthetic phosphopeptides corresponding to residues surrounding Ser79 of rat Acetyl-CoA Carboxylase and Ser491 of human AMPK 2. Monoclonal antibodies are produced by immunizing rabbits with a synthetic phosphopeptide (KLH-coupled) corresponding to residues surrounding Thr172 of human AMPK , the amino-terminal sequence of human AMPK, residues surrounding His231 of AMPK 1 and residues surrounding Ser52 of human Acetyl-CoA Carboxylase 1.

Vol. Applications MW Reactivity A1475-25A: Phospho-AMPK alpha (Thr172) 20ul W, IHC-P 62kD Rabbit Mab H M R Mk Sc Dm (C) (B) A1475-25B: AMPK alpha 20ul W,IP,F 62kD Rabbit Mab H M R Mk A1475-25C: AMPK beta1Phosphorylated (Ser108) 20ul W,IP 38kD Rabbit Pab H M R Mk A1475-25D: AMPK beta1/2 20ul W,IHC-P, IF-IC, F 34,38kD Rabbit Mab H M R Mk Hm A1475-25E:Acetyl-CoA Carboxylase Phosphorylated (Ser79)20ul W, IP, IHC-P, IF-IC 280kD Rabbit Pab H M R Mk (C) (B) A1475-25F: Acetyl-CoA Carboxylase 20ul W, IHC-P, IF-IC, F 280kD Rabbit Mab H M R A1475-25G: IgG (HRP) 100ul Goat Pab

Western Blot IHC-P IP IF-IC F A1475-25A 1:1000 1:50 A1475-25B 1:1000 1:100 1:50 A1475-25C 1:1000 1:50 A1475-25D 1:1000 1:50 1:50 1:100 A1475-25E 1:1000 1:400 1:100 1:25 A1475-25F 1:1000 1:25 1:200 1:400

Optimal dilutions to be determined by the researcher.

May be stored at 4°C for short-term only. For long-term storage, aliquot and store at -20°C. Aliquots are stable for at least 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

Purified by Protein A and peptide affinity chromatography.

Supplied as a liquid in 10mM sodium HEPES, pH 7.5, 150mM sodium chloride, 0.1mg/ml BSA and 50% glycerol.

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.