AMPK Methods and Protocols
3.3 Preparation of the HPLC System The HPLC system used (DIONEX UltiMate 3000, Thermo Sci- entific) consists of a column module ...
module (seeNote 4). A security guard cartridge pre-column (SUPELCOSIL™LC-18-DB Supelguard™Cartridge, 5μm, 2cm 3 mm, Sigma-Aldri ...
as broadening of peaks, consider regenerating the column (go tostep 9). Column storage: Purge the system. Flush the column in 5 ...
where [ATP], [ADP], and [AMP] are the respective intracellu- lar/tissue concentrations inμmol. 4 Notes This is advised to remov ...
Guigas B, Bertrand L, Taleux N, Foretz M, Wiernsperger N, Vertommen D, Andreelli F, Viollet B, Hue L (2006) 5-Aminoimidazole-4- ...
Chapter 16 Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation Simo ...
berberine); (2) directly, by binding at site 3 (e.g., ZMP, derived from intracellular metabolism of the prodrug 5-aminoimidazole ...
[2]. This binding site appears to be stabilized by an ionic interaction between phosphorylated Ser108 onβ1 (which can be modifie ...
that AMPK is very sensitive to stress and can be readily phosphory- lated and activated during harvesting and extraction of cell ...
2.2 Methods of Harvesting Adherent Cells for AMPK Assays or Monitoring Thr172 Phosphorylation Liquid nitrogen. Phosphate-buffer ...
Buffer A: 0.8% (v/v) HCl. Buffer B: 0.1 M NaOH. Buffer C: 50 mM NaH 2 PO 4 , 25 mM NaCl, adding Na 2 HPO 4 to give pH 5.2. Buff ...
1 h in 1% DMSO, may be toxic to the cells and lead to AMPK activation even in vehicle controls. Compounds dissolved in water, cu ...
Gently wash the pellet in 2 ml of ice-cold PBS and repeat centrifugation. Remove PBS by aspiration and immediately resuspend th ...
(b) For a 6 cm plate, add 150μl of ice-cold 5% perchloric acid, scrape off cells using a cell scraper, and transfer the liquid i ...
3.7 Analysis of Cellular Oxygen Uptake Using an Extracellular Flux Analyzer The protocol below assumes the use of a Seahorse XF2 ...
same volume of liquid added. All wells, including the control wells and any blanks, must have vehicle or compound loaded in the ...
covalent modification of Thr172 and allosteric activation and give the most sensitive indication of the degree of AMPK activatio ...
“Slow lysis” is performed at room temperature; this causes cell stress, thus activating AMPK. Aspirate medium, add 500μlof PBS ...
gradient, causes OCR to reach a maximum, as mitochondria are no longer limited by the supply of ADP. However, prior addition of ...
References Li X, Wang L, Zhou XE, Ke J, de Waal PW, Gu X, Tan MH, Wang D, Wu D, Xu HE, Melcher K (2015) Structural basis of AMP ...
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