Anti t ampk

Manufactured by Cell Signaling Technology

Sourced in Belgium

Anti-T-AMPK is a laboratory reagent used to detect and quantify the phosphorylation of the threonine residue on the AMPK (AMP-activated protein kinase) enzyme. AMPK is a crucial cellular energy sensor that plays a pivotal role in the regulation of metabolism. The Anti-T-AMPK product enables researchers to study the activation state of AMPK in various experimental systems.

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Anti-AMPK (214 citations) T-AMPK (9 citations)

4 protocols using anti t ampk

Immunoblotting of Brown Adipose Tissue

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Brown adipose tissues were lysed in RIPA buffer with Complete Protease Inhibitor Cocktail (Roche Inc., Indianapolis, IN). Protein concentration was determined with BCA protein assay kit (Pierce, Rockford, IL). Twenty microgram of protein of each sample was separated by SDS-PAGE, and electro-transferred to nitrocellulose membrane for immunoblot analyses. The following antibodies were used: anti-p-PKA (Tyr197) (Cell Signaling, Danvers, MA, 4781S, 1:1000), anti-t-PKA (Cell Signaling, Danvers, MA, 4782, 1:1000), anti-p-HSL (Ser563) (Cell Signaling, Danvers, MA, 4139S, 1:1000), anti-t-HSL (Cell Signaling, Danvers, MA, 4107, 1:1000), anti-p-Creb (Ser133) (Cell Signaling, Danvers, MA, 9191S, 1:1000), anti-t-Creb (Cell Signaling, Danvers, MA, 9197, 1:1000), p-AMPKα (T172) (Cell Signaling, Danvers, MA, 2535L, 1:1000), anti t-AMPK (Cell Signaling, Danvers, MA, 2603S, 1:1000), anti-UCP1 (ABCAM, Cambridge, MA Ab10983, 1:10000), anti-β-actin (Cell Signaling, Danvers, MA, 4967S, 1:1000), HRP-conjugated anti-mouse (GE Healthcare UK Limited, 1:10,000), and anti-rabbit (GE Healthcare UK Limited, 1:10,000). The SuperSignal West Pico Chemiluminescent kit (Pierce, Rockford, IL) was used as substrates.

Lin L., Lee J.H., Bongmba O.Y., Ma X., Zhu X., Sheikh-Hamad D, & Sun Y. (2014). The suppression of ghrelin signaling mitigates age-associated thermogenic impairment. Aging (Albany NY), 6(12), 1019-1032.

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AMPK Phosphorylation Analysis by Western Blot

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Cell lysates were subjected to SDS-PAGE and western blot analysis. Briefly, cells were lysed with protein lysis buffer (RIPA, Thermo) followed by heat denaturation. Five micrograms of whole cell proteins were applied to SDS-PAGE. After electrophoresis, the proteins were transferred onto PVDF membrane, followed by membrane blocking in TBS-T buffer containing 5% non-fat dry milk for 1 hour at room temperature. The membrane was probed with the following different primary antibodies: anti-phosphorylated-AMPK (Thr172) (Cell Signaling), anti-tAMPK (Cell Signaling), anti-pACC, and anti-GAPDH (Santa-Cruz). Then, the membrane was washed and incubated with peroxidase-conjugated secondary antibody and finally visualized using Chemiluminescent HRP Substrate reagent (Millipore MA, USA) in Gel-Doc imaging system (Bio-Rad). Signals from Western blot were quantified by using Image Lab Software 6.0 (Bio-Rad).

Kim S., Lee Y., Kim J.W., Son Y.J., Ma M.J., Um J.H., Kim N.D., Min S.H., Kim D.I, & Kim B.B. (2018). Discovery of a novel potent peptide agonist to adiponectin receptor 1. PLoS ONE, 13(6), e0199256.

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Puerarin Attenuates LPS-Induced Oxidative Stress

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48 healthy male Sprague-Dawley rats (aged 8-10 weeks) were purchased from Model Animal Research Center. Puerarin was obtained from Sigma-Aldrich (P5555). Lipopolysaccharide (LPS, ST1470) and One Step TUNEL Apoptosis Assay Kit (C1088) were purchased from Beyotime Biotechnology. The following antibodies were used in this study: anti-cleaved caspase3 (Cell Signaling Technology, 9661), anti-Bax (Cell Signaling Technology, 2772), anti-Bcl2 (Cell Signaling Technology, 3498), anti-GPX4 (Cell Signaling Technology, 52455), anti-ACSL4 (Santa Cruz Biotechnology, sc-365230), anti-Ferritin (Abcam, ab75973), anti-TFR (Abcam, ab84036), anti-P-AMPK (Cell Signaling Technology, 50081), anti-T-AMPK (Cell Signaling Technology, 5832), and anti-GADPH (Beyotime Biotechnology, AF0006). Then enzyme-linked immunosorbent assay (ELISA) kit for the detection of IL-6 (H007), IL-10 (H009) and TNF-α (H052) were purchased from Nanjing Jiancheng Bioengineering Institute. In addition, oxidative stress indicators (MDA, GSH, and SOD) were detected by the ELISA kit from Nanjing Jiancheng Bioengineering Institute.

Zhou B., Zhang J., Chen Y., Liu Y., Tang X., Xia P., Yu P, & Yu S. (2022). Puerarin protects against sepsis-induced myocardial injury through AMPK-mediated ferroptosis signaling. Aging (Albany NY), 14(8), 3617-3632.

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Hepatic Protein Expression Analysis

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Hepatic proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After 1 h of blocking with 2% fat-free milk, membranes were incubated overnight with antibodies against CYP2E1, CYP2A5, acyl-CoA oxidase (AOX), alcohol dehydrogenase (ADH), PPARα, fatty acid synthase (FAS), Carnitine palmitoyltransferase I (CPT I), phosphorylated AMP-activated protein kinase (p-AMPK), total AMPK (T-AMPK), and calnexin or β-actin followed by 1 h incubation with peroxidase secondary anti-rabbit, anti-chicken, and anti-goat antibodies (Millipore). Calnexin or β-actin were detected as protein loading control. Anti-CYP2E1 IgG was gifted by Dr. Jerome Lasker, Hackensack Biomedical Research Institute, Hackensack, NJ; anti-CYP2A5 IgG was gifted by Dr. Risto Juvonen, Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland; anti-AOX was gifted by Professor Paul Van Veldhoven, K.U. Leuven, Belgium; finally, anti-p-AMPK and anti-T-AMPK were purchased from Cell Signalling Technology, and the remaining antibodies were from Santa Cruz Biotechnology, CA. Chemiluminescence was detected by Image Reader LAS-4000 (Fijifilm) after adding Pierce EC Western Blotting Substrate (Thermo Scientific). The bands of proteins were quantified with the Automated Digitizing System (ImageJ gel programs, version 1.34S; National Institutes of Health, Bethesda, MD).

Hong F., Liu X., Ward S., Xiong H., Cederbaum A.I, & Lu Y. (2015). Absence of cytochrome P450 2A5 enhances alcohol-induced liver injury in mice. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver, 47(6), 470-477.

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