Primary astrocytes were seeded at 5 × 105 cells/well in 6-well plates, allowed to attach overnight, and then incubated in DMEM containing 1 μg/mL adiponectin for 24 h. The cells were washed twice with PBS, followed by scraping and resuspension of the cell pellet in RIPA lysis buffer containing protease inhibitors and centrifugation to remove debris, unbroken cells, and cellular nuclei. Protein content was determined by Bradford’s method using bovine serum albumin as a standard. Samples containing 10 µg of total protein were separated using 12% sodium dodecyl sulfate acrylamide gels. After electrophoresis, proteins were transferred to a nitrocellulose membrane and then blocked with 5% skim milk in TBS (Tris-buffered saline, 0.1% Tween20) buffer for 2 h and incubated in the primary antibodies, including anti-GFAP (1:1000 dilution, Sigma-Aldrich, USA), anti-Iba-1 (1:1000 dilution, Wako, Osaka, Japan), anti-Glut1 (1:500 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phosphorylated (p)AMPK, anti-total AMPK (1:1000 dilution, Cell Signaling, Danvers, MA, USA), and anti-β-actin (1:10,000 dilution, Sigma-Aldrich, USA).
Anti phosphorylated p ampk
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phosphorylated (p)AMPK is a laboratory reagent used to detect the phosphorylated form of the AMP-activated protein kinase (AMPK) enzyme. AMPK is a key cellular energy sensor that becomes activated upon phosphorylation. This antibody can be used to monitor AMPK activation in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Anti glut1, by Santa Cruz Biotechnology (1 mentions)
Anti total ampk, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Solarbio (1 mentions)
Anti acc, by Cell Signaling Technology (1 mentions)
Ab110413, by ABclonal (1 mentions)
Ripa lysis buffer, by Fdbio (1 mentions)
Anti ampk, by Cell Signaling Technology (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
2 protocols using anti phosphorylated p ampk
1
Adiponectin Effects on Primary Astrocytes
2
Western Blot Analysis of Metabolic Regulators
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Differentiated primary satellite cells in 6-or 12-well plates were quickly lysed in situ in 1x SDS-sample buffer (200 µl/well for 6-well plates or 100 µl/well for 12-well plates), followed by denaturation. For muscle tissue samples, frozen samples (20 mg) were immediately ground on ice in 200 µl RIPA lysis buffer (Fdbio science, #FD008) in the presence of protease inhibitors (Solarbio, P0100). Samples were then homogenized in 5 × SDSsample buffer at a final concentration of 1×, followed by denaturation. Protein samples were run on a 10% SDSpolyacrylamide gel, and Western blot analysis was performed. Primary antibodies were diluted 1:1000, and included anti-phosphorylated (p) AMPK (Cell signaling technology 2535), anti-AMPK (Cell signaling technology 2532), anti-phosphorylated (p) ACC (Cell signaling technology 3661L), anti-ACC (Cell signaling technology 3662), anti-SIRT1 (Abcam, ab110304), anti-CPT1b (Proteintech, 22170-1-AP), anti-CD36 (Abclonal, A19016), anti-PGC1a (Abclonal, A12348), anti-OXPHOS (Abcam, ab110413) and anti-GAPDH (Abclonal, Ac001). GAPDH was used as a loading control. The bands on Western blots were analyzed using Image J.
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