P ampk

Manufactured by Merck Group

Sourced in United States

P-AMPK is a laboratory assay used to measure the phosphorylation of the AMP-activated protein kinase (AMPK) enzyme. AMPK is a key regulator of cellular energy metabolism and acts as a sensor of cellular energy status. The P-AMPK assay provides a quantitative measure of AMPK phosphorylation, which can be used to assess AMPK activation in various cell-based or in vitro experimental systems.

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Lab products found in correlation

β actin, by Merck Group (2 mentions) Pgc 1α, by Abcam (1 mentions) Collagen 2, by Proteintech (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Image lab 3, by Bio-Rad (1 mentions) Mmp13, by Cell Signaling Technology (1 mentions) Sirt3, by Cell Signaling Technology (1 mentions) Aggrecan, by Proteintech (1 mentions) Bnip3, by Abcam (1 mentions) β actin, by Abcam (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Adamts5, by Cell Signaling Technology (1 mentions) Mmp 3, by Cell Signaling Technology (1 mentions) Bovine serum, by Roche (1 mentions) Neurofilament l, by Agilent Technologies (1 mentions) Protease inhibitor, by Thermo Fisher Scientific (1 mentions) Ecl detection reagent, by GE Healthcare (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions) Ripa lysate, by Thermo Fisher Scientific (1 mentions) P mtor, by Merck Group (1 mentions)

Spelling variants of this product

Anti-p-AMPK (5 citations) Anti-p-AMPK antibody (2 citations)

8 protocols using p ampk

Chondrocyte Protein Expression Analysis

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Rat chondrocytes were lysed in ice-cold RIPA with 1 mM PMSF (Phenylmethanesulfonyl fluoride, Beyotime). Protein concentrations of samples were measured by the BCA protein assay kit (Beyotime). Proteins of chondrocytes was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transfferred to polyvinylidene difluoride membrane (Millipore, USA) followed by blocking with 5% nonfat milk. After that, the bands were probed with primary antibodies specific to SIRT3 (1:1000, Cell Signaling Technology), MMP-3 (1:1000, Cell Signaling Technology), MMP-13 (1:1000, Cell Signaling Technology), ADAMTS-5 (1:1000, Cell Signaling Technology), aggrecan (1:1000, Proteintech), collagen II (1:1000, Proteintech), p-AMPK (1:500, Sigma), AMPK (1:1000, Sigma), PGC-1α (1:1000, Abcam), MFN2 (1:1000, Abcam), BNIP3 (1:1000, Abcam), NIX (1:1000, Santa cruz), LC3 (1:1000, Cell Signaling Technology), DRP1 (1:1000, Abcam) , FIS1 (1:1000, Genetex) and β-actin (1:1000, Abcam) overnight at 4°C, before incubated with respective secondary antibodies. Last, the intensity of these bands was quantified using Image Lab 3.0 software (Bio-Rad).

Wang J., Wang K., Huang C., Lin D., Zhou Y., Wu Y., Tian N., Fan P., Pan X., Xu D., Hu J., Zhou Y., Wang X, & Zhang X. (2018). SIRT3 Activation by Dihydromyricetin Suppresses Chondrocytes Degeneration via Maintaining Mitochondrial Homeostasis. International Journal of Biological Sciences, 14(13), 1873-1882.

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Immunostaining of Optic Nerve Sections

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Three eyes 1 week after laser irradiation or three normal eyes were collected and fixed by immersion in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections (4 μm thickness) were made through the optic disc and blocked with 1% bovine serum (Roche Diagnostics GmbH, Mannheim, Germany). The primary antibodies were against p-AMPK (1:100; Sigma-Aldrich) and neurofilament-L (a marker of nerve fibers; 1:100; Dako, Tokyo, Japan). The secondary antibodies were FITC-labeled or rhodamine-labeled antibodies (1:100; Cappel, Aurora, OH, USA). The sections were mounted on slides in a DAPI-containing medium under the cover of glass. The images were captured using a confocal microscopy system (Zen; Carl Zeiss QEC GmbH, Köln, Germany).

Arizono I., Fujita N., Tsukahara C., Sase K., Sekine R., Jujo T., Otsubo M., Tokuda N, & Kitaoka Y. (2023). Axonal Protection by Oral Nicotinamide Riboside Treatment with Upregulated AMPK Phosphorylation in a Rat Glaucomatous Degeneration Model. Current Issues in Molecular Biology, 45(9), 7097-7109.

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Western Blot Analysis of Cardiac Proteins

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To obtain the total proteins, myocardial tissues or cells were lysed with RIPA lysate containing protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The protein was subjected to SDS-PAGE, and then was transferred onto PVDF membrane. The membranes were blocked by nonfat milk and then incubated at the temperature of 4 °C overnight with primary antibodies (Bax, 1:1000, 14,796; Bcl-2, 1:1000, 4228 s, Cell Signaling Technology, Danvers, MA, USA; SERPINB1, 1:1000, sc-293462; caspase-3, 1:1000, sc-271759, Santa Cruz Biotechnology, Santa Cruz, CA, USA; AMPK, 1:1000, SAB4502329, p-AMPK, 1:1000, SAB4503754; mTOR, 1:1000, T2949, p-mTOR, 1:1000, SAB4504476, Sigma Aldrich, St. Louis, MO, USA). Subsequently, membranes were incubated with secondary antibodies (Sigma Aldrich, St. Louis, MO, USA) properly under room temperature. The western blots were visualized with ECL detection reagents (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Wang H., Hua J., Chen S, & Chen Y. (2022). SERPINB1 overexpression protects myocardial damage induced by acute myocardial infarction through AMPK/mTOR pathway. BMC Cardiovascular Disorders, 22, 107.

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Western Blot Analysis of OXPHOS and AMPK

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HepG2 cells were collected in Laemmli buffer and protein samples were transferred to membranes by electrotransfer from sodium dodecyl sulfate polyacrylamide gel (10%) using a Bio-Rad Trans-Blot system (Bio-Rad Laboratories, Inc., Tokyo, Japan). Membranes were blocked for 1 h at room temperature in 5% nonfat milk diluted in Tris buffered saline solution containing 0.1% Tween 20 (TBS-T). Then the membranes were washed in TBS-T and incubated with primary antibodies for OXPHOS, AMPK, p-AMPK and beta-actin (Sigma-Aldrich) overnight, under stirring at 4 °C. After that, the membranes were incubated with secondary antibodies for 2 h at room temperature. Finally, the detection of the blot was realized with a chemiluminescence kit (substrate ECL Western Clarity^TM, Bio-Rad, Hercules, CA, USA).

Oliveira K.A., Araújo H.N., Lima T.I., Oliveira A.G., Favero-Santos B.C., Guimarães D.S., Freitas P.A., Neves R.D., Vasconcelos R.P., Almeida M.G., Ramos M.V., Silveira L.R, & Oliveira A.C. (2021). Phytomodulatory proteins isolated from Calotropis procera latex promote glycemic control by improving hepatic mitochondrial function in HepG2 cells. Saudi Pharmaceutical Journal : SPJ, 29(9), 1061-1069.

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Protein Extraction and Immunoblotting of Tumor Samples

Cited 1 time

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The collected tumor samples were used to prepare specimens. TissueLyzer II (Qiagen) was used for the radioimmunoprecipitation assay (RIPA) buffer-mediated disruption of cells to obtain cell lysates, which were then centrifuged. The Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific) or Bradford Protein Assay Kit (Bio-Rad) was employed to determine concentrations of proteins. Immunoblot assay was used as previously described [20 (link)] in the presence of antibodies against cleaved caspase-3, Bid, Bim, AMPK, p-AMPK, β-actin (Sigma-Aldrich), Mcl-1 (BD Biosciences), Bax, Bad, Bcl-xL, Bcl-2 (Agilent DAKO, USA), PUMA, and p-S6k, S6k, 4EBP1, p-4EBP1, TSC1, TSC2 (Abcam).

Ye H., Liu Y., Wu K., Luo H, & Cui L. (2020). AMPK activation overcomes anti-EGFR antibody resistance induced by KRAS mutation in colorectal cancer. Cell Communication and Signaling : CCS, 18, 115.

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Western Blot Profiling of Cellular Signaling

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Protein samples were prepared by lysing (Cell signaling lysis buffer, #9803) and sonicating (Bandelin Sonopuls, Berlin, Germany) of the cells and quantified according to Bradford’s method68 (link). SDS-PAGE was performed with a 10% (>40 kDa) or 16% (<40 kDa) gel and 30 μg protein extract per lane. The separated proteins were transferred to a polyvinylidene fluoride membrane by semi-dry western blotting, followed by incubation with different antibodies. The following antibodies by Cell Signaling were used with a dilution of 1:1000: p-Akt (#9271), Akt (#9272), p-p70S6K (#9205), p-S6 (#2211), p-4E-BP1 (#9451), p-GSK3αβ (#9331), GSK3β (#9315), cleaved Caspase 3 (#9664), PARP (#9542), p-AMPK (#2531), AMPK (#2532), anti-rabbit (#7074) as well as α-Tubulin by Sigma-Aldrich (#T9026). All protein samples were generated for immunoblot detection in 3 independent experiments.

Poburski D., Leovsky C., Boerner J.B., Szimmtenings L., Ristow M., Glei M, & Thierbach R. (2016). Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model. Scientific Reports, 6, 37120.

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Western Blot Analysis of Mitochondrial Proteins

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Proteins were electrophoresed on 8–12% v/v polyacrylamide SDS-PAGE gels. Proteins were electrotransfered onto PVDF membranes. The membranes were subsequently washed, and after blocking, PVDF membranes were incubated at 4°C with antibodies (1:1000 #sc-69359 Santa Cruz COX-4, 1:150 S7150 Chemicon anti-DNPH, 1:1000 #2459 Cell Signaling PSMA6, 1:500 LonP1, 1:5000 #ab87253 Abcam HSP78, 1:1400 #2532S Cell Signaling AMPK, 1:500 #2535S Cell Signaling pAMPK, 1:500 #07–131 Millipore, Upstate SIRT1, 1:500, sc-13067 Santa Cruz PGC1-α, 1:1000 #sc-30963 Santa Cruz TFAM, 1:1000 #sc-33771 Santa Cruz NRF-1, 1:500 #sc-98900 Santa Cruz Fis1, 1:3000 #50330 Santa Cruz Mfn1, 1:15000 #T6199 Sigma α-tubulin). After incubation with primary antibodies, membranes were washed 3x10 minutes in TBS-Tween-20 (TBS-T) and incubated with horseradish peroxidase (HRP) – conjugated secondary antibodies at 4°C in one hour. After incubation with primary antibodies, membranes were washed in TBS-T and incubated with HRP-conjugated secondary antibodies. After incubation with a secondary antibody, membranes were repeatedly washed. Membranes were incubated with an HRP plus reagent (Super Signal West Pico Chemiluminescent Substrate, Thermo Scientific #34080) and protein bands were visualized on X-ray films. The bands were quantified by ImageJ software, and normalized to α-tubulin, which served as an internal control.

Marton O., Koltai E., Takeda M., Koch L.G., Britton S.L., Davies K.J., Boldogh I, & Radak Z. (2014). Mitochondrial biogenesis-associated factors underlie the magnitude of response to aerobic endurance training in rats. Pflugers Archiv : European journal of physiology, 467(4), 779-788.

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Insulin Signaling in Myotubes

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Myotubes pretreated with or without fatty acids were stimulated with or without 100 nM insulin for 10 min at +37°C. Total and phosphorylated proteins were detected by Western blotting in PVDF membranes with primary antibodies from Cell Signaling: p-Akt^Ser473 (#9271), total Akt (#9272), pAS160^Thr642 (#4288), total AS160 (#2447), p-GSK-3β^Ser9 (#9336), total GSK-3β (#9315), total PERK (#3192), p-JNK^{Thr183/Tyr185} (#9251), total JNK (#9252), total AMPK (#2532), from Santa Cruz: p-PERK^Thr981 (#sc-32577) and from Millipore: p-AMPK (#07-626). Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibody, visualized by enchanced chemiluminescence (Pierce ECL 2 Western Blotting Substrate, Thermo Scientific) and quantified using ImageJ software (NIH, http://rsbweb.nih.gov/ij/).

Mäkinen S., Nguyen Y.H., Skrobuk P, & Koistinen H.A. (2017). Palmitate and oleate exert differential effects on insulin signalling and glucose uptake in human skeletal muscle cells. Endocrine Connections, 6(5), 331-339.

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