AMPK Methods and Protocols

The 6SB should comprise a sixth of the total volume in a sample made ready for SDS-PAGE. 170μl Post-IP/5¼ 34 μl. Leave a small ...

role for gamma3 in white skeletal muscle. Am J Physiol Endocrinol Metab 286:E194–E200 Yu H, Fujii H, Hirshman MF, Pomerleau JM, ...

Chapter 14 Kinase Activity Determination of Specific AMPK Complexes/ Heterotrimers in the Skeletal Muscle Jesper B. Birk and Jør ...

(ACC). This peptide was modified to omit phosphorylation by cyclic AMP-dependent protein kinase and called the SAMS peptide from ...

heterotrimeric complex. This has previously been shown for the α 2 β 2 γ3 complex by correlating theγ3-associated activity to th ...

Reusable silicone sealing mat for PCR-plate(s) (Fig.1A). Homemade “sandwich device” to keep the silicone sealing mat firmly in ...

96-Well aluminum block for the PCR-plate to keep it cold on ice during the IP procedure and warm on a heating block during the ...

including blind samples (seestep 6) and three consecutive IPs for measuring the activities ofα 2 β 2 γ3,α 2 β 2 γ1, andα 1 β 2 γ ...

When all supernatants are transferred, seal the “α2” PCR-plate and install it to rotate overnight at 4C as described for the “ ...

When the kinase reaction has run for 30 min, remove the PCR-plate from the heating block and stop the reaction by adding 10μl o ...

Subheading3.2), a relationship between “volume” and DPM is known. Dividing the “volume” with the DPM for the 10μlof the SA gives ...

4 Notes The IP-buffer could be the same as the homogenization buffer or even just phosphate-buffered saline (PBS). The importan ...

washing, then this is a total of (120μlþ 540 μl) * 100¼66 ml of IP-buffer. Approximately 10 ml of IP-buffer will be used for the ...

The time and temperature for an IP can vary a lot depending on antibody affinity, considerations for protein complex stability, ...

calibration date for the latter. If the assay is run after the calibration date, then more of the^33 P-ATP stock is needed and a ...

analyzed by liquid scintillation by putting them into counting vials with Ultima Gold. In this case the filter paper will “quenc ...

Chapter 15 Determination of Adenine Nucleotide Concentrations in Cells and Tissues by High-Performance Liquid Chromatography Noe ...

inhibition of Thr172 dephosphorylation by still unidentified AMPK-specific protein phosphatase(s) [4]. Once activated, AMPK conc ...

KOH/MOPS solution: 2 N KOH/0.3 M 3-(N-morpholino) propanesulfonic acid (MOPS). Add ~70 ml of mQ water to a glass beaker contain ...

Weigh 9.1 mg of AMP monohydrate and transfer to the glass beaker container. Mix and adjust pH to 7.4 with Tris–HCl powder. Make ...