AMPK Methods and Protocols
Chapter 5 Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK Fiona A. Fyffe, Simon A. Hawley, Alexander Gray, and ...
(2) promotion of phosphorylation at Thr172 by upstream kinase (s) via binding of either AMP or ADP, and (3) inhibition of dephos ...
In this chapter, we will discuss how cell-free assays can be used to test the effects of ligands and drugs on the three mechanis ...
(weigh 250 g glycerol, 25 g glucose, 100 gα-lactose, gently heated and dissolved in 730 mL deionized water and auto- claved at 1 ...
Synthetic peptide substrate [either SAMS (HMRSAMSGLHLVKRR [21]) or AMARA (AMARAA- SAAALARRR[22])], 1 mM in kinase assay buffer. ...
2.7 Promotion of Thr172 Phosphorylation AMPK, either immunoprecipitated from mammalian cells or purified from bacterial cells i ...
Add small amounts of the ATP to the buffer, allowing each addition to fully dissolve before adding more. Maintain the pH betwee ...
also be used, depending on protein recovery and other require- ments. Recover cells by centrifugation at 7000gfor 30 min at 4C ...
Having decided how much CaMKK2 to use to obtain maximal Thr172 phosphorylation, scale up the activation assay with the required ...
Place each square into a scintillation vial containing 3 mL of scintillation fluid and count them using a suitable^32 P program ...
followed by 21 mL of IP buffer. Resuspend the final pellet in 1 mL total volume of IP buffer and divide into 20 50 μL aliquots ...
substrate, because (for reasons that remain unclear) a higher AMP dependence is always obtained using the former. With any new a ...
3 – 4 below refer to in-solution and IP kinase assays, respectively. Step 5is common to both methods. For in-solution assays, m ...
For immunoprecipitation assays mix the following components in 1.5 mL microcentrifuge tubes: (1) 20μL of AMPK immu- noprecipita ...
immunoprecipitated from a cell line that expresses LKB1 (HEK293, COS7). Before lysis, the user may treat the cells with an activ ...
A549). If material is active, the user must dephosphorylate the starting material; we recommend using PP1γand then using okadaic ...
Structural basis for AMP binding to mamma- lian AMP-activated protein kinase. Nature 449 (7161):496–500 Chen L, Wang J, Zhang Y ...
dependent protein kinase I. FEBS Lett 361:191–195 Ross FA, Rafferty JN, Dallas ML, Ogunbayo O, Ikematsu N, McClafferty H, Tian ...
Chapter 6 Applications of NMR and ITC for the Study of the Kinetics of Carbohydrate Binding by AMPKβ-Subunit Carbohydrate-Bindin ...
shown for other proteins [5], combining isothermal titration calo- rimetry (ITC) and various nuclear magnetic resonance (NMR) sp ...
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