AMPK Methods and Protocols
of the colonies after periods of 30 min. By comparison with the positive and negative control, one can estimate qualitatively th ...
The units ofβ-galactosidase activity (Miller Units) are defined as value of A 420 2000/time of the reaction in minutes [24]. ...
Once the best ratio of the combination of bait and cDNA library plasmid is determined, repeat the transformation under these co ...
medium lacking only leucine, since the prey plasmid contains a LEU2selection marker. At the exponential phase (A 600 0.5) remove ...
strength of the interaction is normally higher when using a LexA-based bait plasmid in comparison to the GBD-based one and, in a ...
AMPKγ3 has a strong self-activating profile [13]. In the case of weak self-activating properties, the regular measurement of the ...
they are heat shock, ribosomal and mitochondria proteins; proteasome subunits, elongation factors, etc. The collection of positi ...
M (2014) A proteome-scale map of the human interactome network. Cell 159(5):1212–1226 Viana R, Towler MC, Pan DA, Carling D, Vi ...
Chapter 10 Transient Expression of AMPK Heterotrimer Complexes in Mammalian Cells Jonathan S. Oakhill, John W. Scott, and Toby A ...
potentially permit formation of 12 distinct AMPK complexes (α 1 β 1 γ1 through α 2 β 2 γ3). Much of the pioneering work on AMPK ...
2 Materials 2.1 Expression of Recombinant AMPK Expression constructs required for the method are described below (seeNotes 1and ...
γ2 (Q9UGJ0), orγ3 (Q9UGI9) were inserted into the cloning site using restriction sites PstI and EcoRI. Addition of HA affinity t ...
CaMKK2 phosphorylation buffer: 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 10% (v/v) glycerol, 2 mM MgCl 2 , 200 μM ATP. 7.λ ...
Aspirate PBS and add 0.5 ml of ice-cold reduced cell lysis buffer to the top edge of the dish. Using a cell scraper, collect so ...
3.3 Modification of the Protocol for Postharvest Increase or Decrease of AMPK Activity We find that recombinant AMPK extracted f ...
Wash anti-FLAG M2 affinity agarose gel three times by resus- pension in 1 ml of FLAG agarose wash buffer, followed by centrifug ...
Pellet glutathione agarose by centrifugation at 3,350gfor 3 min. Wash glutathione agarose three times by resuspension in 1 ml ...
from Promega; however alternative transfection reagents from other suppliers can be used and their use optimized empirically. W ...
purified AMPK can be eluted from anti-FLAG M2 affinity agarose gel using FLAG elution buffer instead of glutathione agarose elut ...
Chapter 11 Knockdown of Human AMPK Using the CRISPR/Cas9 Genome-Editing System Adrien Grenier, Pierre Sujobert, Se ́verine Olivi ...
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