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ab151280 is an In-Cell ELISA (ICE) assay kit that uses quantitative immunocytochemistry to measure levels of total AMPK alpha1 subunit and phosphorylated at threonine 172 AMPK alpha1 subunit in cultured cells.
In-Cell ELISA (ICE) technology is used to perform quantitative immunocytochemistry of cultured cells with a near-infrared fluorescent dye-labeled detector antibody. The technique generates quantitative data with specificity similar to Western blotting, but with much greater quantitative precision and higher throughput due to the greater dynamic range and linearity of direct fluorescence detection and the ability to run up to 96 samples in parallel. This method rapidly fixes the cells in situ, stabilizing the in vivo levels of proteins and their post-translational modifications, and thus essentially eliminates changes during sample handling, such as preparation of protein extracts. Finally, the signal can be normalized to cell amount, measured by the provided Janus Green whole cell stain, to further increase the assay precision.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
AMP-activated protein kinase (AMPK) is an energy sensor protein kinase that plays a key role in regulating cellular energy homeostasis. Mammalian AMPK is a heterotrimer kinase, containing a catalytic subunit (α) and two regulatory subunits (β and γ). Each subunit has different isoforms (α1, α2, β1, β2, γ1, γ2, γ3) with differential tissue expression, cellular localization and functionality. It has been hypothesized that when ADP or AMP are present at high levels, these nucleotides bind directly to the γ subunit, leading to a conformational change that allows phosphorylation of Thr172 at the α subunit. Phosphorylation of AMPK α activates the kinase which leads to downstream effects concerted to increase catabolic and suppress anabolic pathways in order to restore levels of cellular ATP and ultimately cell fate. AMPK is activated physiologically due to stresses such as low nutrients and prolonged exercise. Furthermore AMPK may be activated pharmacologically by metformin (the most widely prescribed Type 2 diabetes drug), phenformin, AICAR (acadesine/AICA riboside) and resveratrol.
|Components||1 x 96 tests|
|100X AMPK (total and pT172/183) Primary Antibody Cocktail||1 x 120µl|
|10X Blocking Solution||1 x 15ml|
|10X Phosphate Buffered Saline||1 x 100ml|
|33x Triton X-100||1 x 1.5ml|
|400X Tween-20||1 x 2ml|
|500X IRDye®-Labeled Secondary Antibody Cocktail||1 x 30µl|
|Antigen Retrieval Buffer||1 x 25ml|
|1X Janus Green Stain||1 x 17ml|
Our Abpromise guarantee covers the use of ab151280 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||Use at an assay dependent concentration.|
ab151280 has not yet been referenced specifically in any publications.