AMPK, ACC, Antibody Sampler Kit, BioAssay(TM)

The AMPK and ACC Antibody Sampler Kit provides an economical means to investigate energy homeostasis and fatty acid synthesis within the cell. The kit contains primary and secondary antibodies to perform four Western mini-blots with each antibody. 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). Kit Components: Vol. Applications MW Reactivity A1475-25A: Phospho-AMPK alpha (Thr172) 40ul W, IHC-P 62kD Rabbit Mab H M R Mk Sc Dm (C) (B) A1475-25B: AMPK alpha 40ul W,IP,F 62kD Rabbit Mab H M R Mk A1475-25C: AMPK beta1Phosphorylated (Ser108) 40ul W,IP 38kD Rabbit Pab H M R Mk A1475-25D: AMPK beta1/2 40ul W,IHC-P, IF-IC, F 34,38kD Rabbit Mab H M R Mk Hm A1475-25E:Acetyl-CoA Carboxylase Phosphorylated (Ser79)40ul W, IP, IHC-P, IF-IC 280kD Rabbit Pab H M R Mk (C) (B) A1475-25F: Acetyl-CoA Carboxylase 40ul W, IHC-P, IF-IC, F 280kD Rabbit Mab H M R A1475-25G: IgG (HRP) 100ul Goat Pab Recommended Dilution: 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. Storage and Stability: May be stored at 4 degrees C for short-term only. For long-term storage, aliquot and store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees 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.