AMPK Methods and Protocols
Day 3: To prepare the inoculum for the 2 L flasks, add 5 mL of LB broth containing 100μg/mL ampicillin to an agar plate that ha ...
3.3 Purification of AMPK Protein This is a 3-day process including some longer 8–9 h days in the laboratory, assuming everything ...
now contains unbroken cells and cell debris should be signifi- cantly smaller. (SeeNote 2.) Filter the supernatant on ice (or i ...
with InstantBlue. After only a few minutes, bands demonstrat- ing the presence of AMPK appear (seeNote 4). Concentrate fraction ...
using a peristaltic pump. This may take some time as the sample is now viscous. Once the entire sample has been applied, attach ...
3.4.2 Re-Optimization of Crystallization Conditions for a New Batch of Protein For a new batch of protein, slight re-optimizatio ...
For a new batch of protein of unphosphorylated protein, the optimal condition for crystallization is likely to have also change ...
The 30 kDa β-subunit has an anomalous electrophoretic mobility. This results in theβ- and theγ-subunits (37 kDa) being difficul ...
SR, Carling D, Gamblin SJ (2013) Structural basis of AMPK regulation by small molecule activators. Nat Commun 4:3017 Calabrese ...
Chapter 2 Visualizing AMPK Drug Binding Sites Through Crystallization of Full-Length Phosphorylatedα 2 β 1 γ 1 Heterotrimer Chri ...
fragment of theβ-subunit [3], the carbohydrate binding module (CBM), and a truncated kinase domain from the orthologous yeast ki ...
laboratories have utilized tricistronic expression vectors to produce recombinant enzyme for full-length AMPK crystallization [6 ...
C2-bound AMPK to co-crystallize [10]. (2) Structural analysis of the α1-RIM reveals it forms a stronger interaction with the γ-s ...
Heating/cooling shaking incubator. IPTG 1000stock: 500 mM isopropylβ-D-1-thiogalactopyr- anoside (IPTG) in water. Spectrophoto ...
2.3 Crystallization for ADaM Site Activators AMP: 9 mM adenosine monophosphate (AMP) solubilized in 50 mM Tris–HCl, pH 7.4. A76 ...
3 Methods 3.1 Expression and Purification of AMPK for Crystallization Transformation of expression vectors: Add 50 ng of either ...
Pass clarified lysate over the nickel column using a peristaltic pump at 1 ml/min. Wash the nickel column with lysis buffer unt ...
Wash FLAG monoclonal antibody-coupled affinity resin exten- sively in insect lysis buffer (510 volumes). Elute CaMKK2 in insec ...
3.5 Cryoprotection of AMPK Crystals Perform the following steps in a 4C cold room. Quickly transfer a single crystal into a 2– ...
4 Notes Levels of AMPK phosphorylation induced by CaMKK2 are critical for producing protein that will crystallize; high levels ...
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