AMPK Methods and Protocols
Macro-seeding is a common crystallographic technique used to improve crystal size. Transfer a crystal into a fresh drop con- ta ...
Kurumbail RG, Calabrese MF (2016) Struc- ture and regulation of AMPK. EXS 107:3–22. https://doi.org/10.1007/978-3-319-43589- 3_ ...
Chapter 3 Biophysical Interactions of Direct AMPK Activators Ravi G. Kurumbail, Graham M. West, Venkatasubramanian Dharmarajan, ...
Modulation of enzymatic activity of proteins by small molecules can be probed by classical enzymology methods which reveal insig ...
complicated by the need for labeled protein reagents. Protein func- tion is governed by local and global structural fluctuations ...
experience this assay is preferred for enzyme kinetic experiments since it can be easily adapted for testing at a range of pepti ...
optimization for expression inSpodoptera frugiperda(Sf-9) insect cells (seeNote 3). Expression media and reagents: LB broth, LB ...
HDX Mobile phase B: 0.3% (v/v) formic acid in acetonitrile: H 2 O (ratio of 4:1). HDX Mobile phase C (protease column buffer): ...
Wash buffer: 50 mM HEPES-NaOH pH 7.4, 1 mM EGTA, 10 mM MgCl 2 , 0.1% (v/v) Tween 20, 0.01% (w/v) BSA. Kinase assay reagents: 10 ...
Fig. 1AMPK expression constructs in bacteria and insect cells. (a) Diagram of recombinant human AMPK heterotrimeric construct in ...
Induce protein expression with 100μM (final concentration) of isopropyl β-D-thiogalactopyranoside (IPTG), reduce the temperatur ...
Prepare fresh H 2 O and D 2 O HDX buffers to match the AMPK protein storage buffer (seeNotes 15and 16 ). Prepare 125μl of each ...
Electrospray ionization parameters are set as follows: sheath gas 30 au, auxiliary gas 15 au, spray voltage 3.7 V, capillary te ...
Initiate each exchange reaction by incubating 4μl of AMPK protein (with or without ligand) with 16μl of AMPK D 2 O buffer for a ...
required for the complete HDX-MS profile analysis of AMPK (42 total injections for a single pairwise comparison for Apo AMPK vs. ...
Sample may begin to show slight precipitate. Spin in a bench- top microcentrifuge ~15,000gfor 10 min at 4C. Set crystal tray ...
1–3 days and typically reach full size by 7–10 days. Wells often become a bit gummy beyond 2 weeks, and crystals beyond this age ...
Solve the crystal structure of AMPKxtalby molecular replace- ment using known structures of different AMPK fragments using the ...
Kmvalue for the specific isoform and to a suggested final^33 P- ATP specific activity of 0.25μCi/nmol (seeNote 40). Since this s ...
3.5 Surface Plasmon Resonance (SPR) Design a suitable SPR construct for the immobilization of AMPK on streptavidin chips(seeNote ...
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