Cells were disrupted by lysis buffer and protein content was determined using the BCA Protein Assay Kit. The quantified protein samples were then separated by SDS-PAGE and transferred to PVDF membranes, followed by sealing with 5% skim milk for 1 hour at room temperature. The PVDF membranes carrying the samples were incubated with primary antibodies overnight at 4°C, and then with secondary antibody for 2 h at room temperature. The signal was displayed with an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.). And, the protein expression levels were semi-quantified using Image-Pro Plus version 6.0 (Media Cybernetics, Inc.) software. The antibodies used in this study were purchased from Abcam and were used at the following concentrations: anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-cleaved caspase3 (1:100), anti-caspase3 (1:500), anti-PDE3B (1:2000), anti-p-AMPK (1:1000), anti-AMPK (1:1000), anti-p-mTOR (1:1000), anti-mTOR (1:10,000), anti-GAPDH (1:2500), and goat anti-rabbit IgG H&L (HRP) (1:2000).
Anti ampk
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-AMPK is a primary antibody that recognizes the AMPK (AMP-activated protein kinase) protein. AMPK is a key cellular energy sensor that plays a crucial role in regulating cellular metabolism and energy homeostasis. This antibody can be used to detect and study the AMPK protein in various applications, such as western blotting, immunohistochemistry, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Anti p ampk, by Abcam (8 mentions)
Anti gapdh, by Abcam (4 mentions)
Anti β actin, by Abcam (4 mentions)
Anti mtor, by Abcam (3 mentions)
Anti phospho ampk, by Abcam (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti sirt1, by Abcam (3 mentions)
Anti caspase 3, by Abcam (2 mentions)
Anti p mtor, by Abcam (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti lpl, by Abcam (2 mentions)
Anti bax, by Abcam (1 mentions)
Image pro plus version 6, by Media Cybernetics (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by HiMedia (1 mentions)
Anti il 6, by Abcam (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Anti nox4, by Abcam (1 mentions)
21 protocols using anti ampk
1
Protein Expression Analysis via Western Blot
2
Western Blot Analysis of Hepatic Proteins
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The hepatic tissue was homogenized into cold lysis buffer and the lysate was centrifuged and the supernatant was collected in a fresh tube supplemented with protease inhibitor cocktail (Himedia, India) and kept at −80 °C.32 The protein samples were heat denatured and separated by SDS-PAGE followed by electrotransfer to the nitrocellulose membrane. The membranes were blocked in blocking buffer and then exposed to specific primary antibodies diluted in the blocking buffer and kept overnight at 4 °C. The primary antibodies used were rabbit polyclonal anti-phospho AMPK, anti-AMPK, anti-IL-6 and anti-TNF-α (Abcam, USA). The next day membrane was washed and subjected to incubation for 1 h with anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (Thermo Fisher Scientific, USA) diluted in the same blocking buffer. After a series of washes, they were exposed with the chemiluminescent ECL substrates (Thermo Scientific, USA) and the desired protein bands were detected by chemiluminescent gel documentation system (Bio-Rad, USA). β-Actin was used as a housekeeping protein for equal amounts of protein loaded into the gel. The protein bands were densitometry quantified by Image J, software (NIH, USA) and data presented as arbitrary units.
3
Protein Expression Analysis via Western Blot
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After cells or tissues were lysed, we used a BCA Protein Assay Kit (Boster Bio, USA) to determine the protein concentration. Total protein was separated by SDS-PAGE. We used a constant current to transfer the separated protein to a PVDF membrane. 5% skim milk was applied to block non-specific reaction sites for 1-2 h. Dilute the primary antibody at the concentration recommended by the instructions, immerse the membrane in this liquid, and place at 4° C for at least 8 hours. The primary antibodies: anti-GAPDH (Proteintech, Wuhan, China), anti-p-ERK (CST), anti-ERK (CST), anti-NOX4 (Abcam, Cambridge, UK), anti-p-AMPK (Abcam), anti-AMPK (Abcam). Horseradish peroxidase-conjugated secondary antibodies were diluted at the concentrations recommended by the instructions, and the membranes were incubated for 2h at room temperature. Enhanced chemiluminescence reagent was used to visualize the protein bands in an imaging densitometer (GE, USA).
4
Detecting AMPK and pAMPK Levels
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Protein levels of AMPK and pAMPK in spinal cord tissue and PC 12 cells were detected by Western-blot analysis. BCA kit (Thermo Scientific) was used to determine protein concentration of each sample. Each band contains a total protein of 40 μg. After electrophoresis and membrane transferring, blots were blocked with milk for 2 h and then incubated with primary antibodies (anti-pAMPK 1:1,000 and anti-AMPK 1:1,000, purchased from abcam; anti-β-actin 1:1,000, purchased from santa cruz). After being washed and incubated with secondary antibodies, blots were imaged by fluorescence scanner (LI-COR Biotechnology, South San Francisco, CA, USA) and data were analyzed.
5
Protein Expression Analysis in fMSCs, hGCs, and Ovarian Tissue
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fMSCs, hGCs, and ovarian tissues were harvested for protein extraction. Western blotting was performed as previously described [12 (link)]. The primary antibodies used for fMSCs were anti-Oct4, anti-Nanog, anti-Rex1, and anti-β-Actin, all purchased from Abcam (USA). The primary antibodies used for hGCs and the ovarian tissues were anti-SURVIVIN, anti-BCL2, anti-CASPASE-3, anti-CASPASE-9, anti-MT1, anti-JNK1, anti-PCNA, anti-AMPK, anti-β-Actin, and anti-GAPDH, all purchased from Abcam (USA).
6
Aortic Protein Expression Analysis
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Proteins (60 µg) extracted from aortas or VSMCs were separated by electrophoresis on
10% polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific
binding sites were blocked with 5% skim milk in Tris-buffered saline solution with
10% Tween for 1 h at 24°C. Membranes were then incubated with antibodies overnight at
4°C. Anti-O-GlcNAc (CTD 110.6, 1:2000; Pierce Biotechnology, USA), anti-AMPK (#80039,
1:1000; Abcam, USA), anti-protein kinase CPI-17 (#32213, 1:1000; Abcam, USA),
anti-MYPT-1 (#2634), anti-rho-kinase (ROCK)-α (#8236), anti-ROCK-β (#4035), anti-MLC
(#8505) and anti-RhoA (#2117) (all 1:1000; Cell Signaling, USA, or BD Biosciences
Transduction Laboratories, USA) were used. Immunoblots for nonphosphoproteins were
carried out on the same membranes used to evaluate the phosphorylated (phospho-)
forms: phospho-MYPT-1 (Thr853), phospho-CPI-17 (Thr38),
phospho-MLC (Thr18/Ser19), and phospho-AMPK
(Thr172), (1:500; Cell Signaling, USA). After incubation with secondary
antibodies, signals were developed for chemiluminescence, visualized by
autoradiography, and quantified densitometrically. Results were normalized to
beta-actin protein (#A5316, 1:10000; Sigma-Aldrich, Inc., USA), or to the total form
of each phosphorylated protein, and reported as arbitrary units.
10% polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific
binding sites were blocked with 5% skim milk in Tris-buffered saline solution with
10% Tween for 1 h at 24°C. Membranes were then incubated with antibodies overnight at
4°C. Anti-O-GlcNAc (CTD 110.6, 1:2000; Pierce Biotechnology, USA), anti-AMPK (#80039,
1:1000; Abcam, USA), anti-protein kinase CPI-17 (#32213, 1:1000; Abcam, USA),
anti-MYPT-1 (#2634), anti-rho-kinase (ROCK)-α (#8236), anti-ROCK-β (#4035), anti-MLC
(#8505) and anti-RhoA (#2117) (all 1:1000; Cell Signaling, USA, or BD Biosciences
Transduction Laboratories, USA) were used. Immunoblots for nonphosphoproteins were
carried out on the same membranes used to evaluate the phosphorylated (phospho-)
forms: phospho-MYPT-1 (Thr853), phospho-CPI-17 (Thr38),
phospho-MLC (Thr18/Ser19), and phospho-AMPK
(Thr172), (1:500; Cell Signaling, USA). After incubation with secondary
antibodies, signals were developed for chemiluminescence, visualized by
autoradiography, and quantified densitometrically. Results were normalized to
beta-actin protein (#A5316, 1:10000; Sigma-Aldrich, Inc., USA), or to the total form
of each phosphorylated protein, and reported as arbitrary units.
7
Western Blot Analysis of AMPK Pathway
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For western blotting analysis, tissue samples were homogenized in ice-cold cell lysis buffer (Beyotime, Shanghai, China) and the lysates were centrifuged at 12,000×g for 20 min at 4°C. The protein concentrations were determined by bicinchoninic acid (BCA) assay (Sigma-Aldrich, St. Louis, MO, USA). The protein samples were then subjected to gel electrophoresis and quantitative western blotting. The following antibodies were used: anti-AMPK, anti-p-AMPKT172, anti-acetyl-CoA carboxylase (ACC), anti-p-ACCS79, and anti-β-Actin (Abcam, Cambridge, UK). The specific bands were detected with an enhanced chemiluminescence detection system (Sage Creation, Beijing, China).
8
Signaling pathway antibody analysis
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Anti-phospho-PAK and anti-phospho-paxillin antibodies were purchased from Millipore (Billerica, MA, USA). Anti-phospho-AMPK and anti-AMPK, and anti-PAK antibodies were purchased from Abcam (Cambridge, UK, USA). Anti-Vav3 and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Metformin and AICAR were obtained from Calbiochem (San Diego, CA, USA).
9
Western Blot Analysis of hADSC Treated with Linoleic Acid
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Briefly, hADSC were treated with 25 μmol LA or Ctrl for 24 h and 48 h (or 80 h) then lysed using RIPA buffer containing protease and phosphatase inhibitors at 4° C, followed by centrifugation for 10 min. The supernatant was collected and sample buffer (5×) was added at a ratio of 5:1. Samples were mixed well and boiled for 10 min, followed by storage at −40° C. Proteins were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes that were blocked by incubation with 2.5% dry skim milk, followed by overnight incubation with primary antibodies diluted in 2.5% dry skim milk at 4° C.
The following antibodies were used: monoclonal rabbit anti-RUNX2, anti-LPL, anti-P16ink4a, anti-GAPDH, anti-MMP14, anti-PKM, anti-PFKP, anti-AMPK, anti-p- AMPK (Abcam) at 1:1000 dilutions. The blots were then incubated with the secondary mouse or rabbit antibodies at room temperature for 1 h. Proteins were detected using the BioSpectrum 600 system. The western blots repeated 3 independent experiments.
The following antibodies were used: monoclonal rabbit anti-RUNX2, anti-LPL, anti-P16ink4a, anti-GAPDH, anti-MMP14, anti-PKM, anti-PFKP, anti-AMPK, anti-p- AMPK (Abcam) at 1:1000 dilutions. The blots were then incubated with the secondary mouse or rabbit antibodies at room temperature for 1 h. Proteins were detected using the BioSpectrum 600 system. The western blots repeated 3 independent experiments.
10
Brain Protein Expression Analysis
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The expression levels of APN, APR1, APPL1, AMPK, pAMPK, FOXO1, and pFOXO1 in the brain and primary neurons were analyzed using Western blot analysis. Proteins from the brain tissues around the infarct area and primary neurons were collected using a protein extraction kit (Epizyme). Bicinchoninic acid assay was used to detect the concentration of proteins. An equal number of proteins in different groups were separated by Tris–glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Consequently, the membranes were blocked with 5% nonfat milk in PBST at room temperature for 1 h and incubated with primary antibodies (anti-APN, Abcam; anti-APR1, Proteintech; anti-APPL1, Proteintech; anti-AMPK, Abcam; anti-pAMPK, Abcam; anti-FOXO1A, Abcam; anti-pFOXO1A, Abcam; and anti-ACTB antibody, Sigma) at 4°C overnight. On the following day, the membranes were incubated with corresponding HRP-conjugated secondary antibody (Abcam) at room temperature for 1 h. The protein band was scanned on Amersham Imager 600 using an enhanced chemiluminescence kit (Sangon Biotech (Shanghai) Co.), and the relative amounts of proteins were analyzed using ImageJ software.
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