Human ORS and DP cells or skin tissue were lysed (RIPA lysis buffer; #20‐188; Merck Millipore). The obtained protein was then separated by 8% SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Amersham). The protein transferred membranes were incubated overnight with the specific antibodies. The following antibodies were used: β‐actin (#MA5‐15739, 1:5,000; Thermo Fisher), phospho‐ERK1/2 (#9101, 1:1,000; Cell Signaling), total ERK1/2 (#9102, 1:1,000; Cell Signaling), phospho‐AMPK (#2535, 1:1,000; Cell Signaling), and total AMPK (#2532, 1:1,000; Cell Signaling). Then, the membranes were washed and incubated with anti‐rabbit IgG or anti‐mouse IgG antibodies (horseradish peroxidase‐conjugated, GTX213110, GTX213111, 1:10,000; GeneTex) at 25°C for 1 h. Antibody‐antigen complexes detected by enhanced chemiluminescent substrate (Thermo Fisher) were captured and quantified by Amersham imager 680 systems (GE Healthcare).
Total ampk
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Total AMPK is a laboratory assay kit that measures the total amount of AMP-activated protein kinase (AMPK) in cellular samples. AMPK is a critical energy sensor that regulates cellular metabolism in response to changes in the AMP/ATP ratio. The Total AMPK assay provides a quantitative determination of AMPK levels, which is useful for studying AMPK signaling pathways.
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Lab products found in correlation
Phospho ampk, by Cell Signaling Technology (15 mentions)
Phospho ampk thr172, by Cell Signaling Technology (9 mentions)
P ampk, by Cell Signaling Technology (8 mentions)
Total akt, by Cell Signaling Technology (8 mentions)
β actin, by Merck Group (8 mentions)
Total acc, by Cell Signaling Technology (7 mentions)
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
Pgc 1α, by Novus Biologicals (5 mentions)
Phospho acc, by Cell Signaling Technology (4 mentions)
Sirt1, by Cell Signaling Technology (4 mentions)
Phospho s6, by Cell Signaling Technology (3 mentions)
Phospho akt, by Cell Signaling Technology (3 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (3 mentions)
Total mtor, by Cell Signaling Technology (3 mentions)
Pthr172 ampk, by Cell Signaling Technology (3 mentions)
γh2ax, by Cell Signaling Technology (2 mentions)
Total cad, by Cell Signaling Technology (2 mentions)
Phospho cad, by Cell Signaling Technology (2 mentions)
Creb 1, by Santa Cruz Biotechnology (2 mentions)
Phospho 4ebp1, by Cell Signaling Technology (2 mentions)
49 protocols using total ampk
1
Western Blot Analysis of Protein Signaling
2
Western Blot Analysis of Protein Signaling
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Protein lysates were prepared in either RIPA or CHAPS buffer and quantified using the BCA Protein Assay (Thermo Scientific). Protein was separated on 4%–20% SDS-PAGE gels, transferred to PVDF membranes, and probed with antibodies against CPS1 (ab3682), β-actin (ab8227), ASS1 (clone 2B10, ab124465), cyclophilin B (clone EPR12703(B), ab178397), total AMPK (Cell Signaling, #2603), phospho-AMPK (Cell signaling, #2531), total ACC (Cell Signaling, #3662), phospho-ACC (Cell Signaling, #11818), LKB1 (Cell Signaling, #3050), γH2AX (Cell Signaling, #9718), total CAD (Cell Signaling, #11933), phospho-CAD (Cell Signaling, #12662), NOS3 (BD, 610298), phospho-S6 (Cell Signaling, #2211), phospho-4E-BP1 (Cell Signaling, #2855), CREB1 (Santa Cruz, sc-186X), FOXA1 (ab23738), TEAD4 (ab58310).
3
Analyzing Cell Signaling Pathways
4
PDAC Cell Protein Quantification
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For the assessment of STAT3, AMPK, or ACC level and phosphorylation status, total cell protein extracts were obtained from human and mouse PDAC cell lines, as well as from mouse tissue samples, via lysis and sonication in RIPA buffer (Cell Signaling, 9806). Total cell lysates were resolved by a 7.5% SDS-PAGE and probed with phospho-STAT3 (Cell Signaling, 9145S), phospho-AMPK (Cell Signaling, 2535S), phospho-ACC (Cell Signaling, 11818T), total STAT3 (Cell Signaling, 9139S) total AMPK (Cell Signaling, 5832S), and total ACC (Cell Signaling, 3676T). To determine equal loading, control membranes were probed with β-actin (Abgent, AM1829B). Quantitation of protein relative amounts of triplicate experiments were analyzed by ImageJ software as a ratio of each phospho protein band relative to the correspondent lane total protein band, followed by ratio to the lane’s loading control.
5
Protein Extraction and Western Blotting
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For protein extraction, tissue was homogenized in RIPA buffer containing protease and phosphatase inhibitor cocktail (#A32959, Thermo Scientific) and protein concentration was measured using the DC™ Protein Assay Reagent (#500‐0114, BioRad). The following primary antibodies were used: phospho‐AMPK (Thr172) (#2531, Cell Signaling Technology), total AMPK (#2603, Cell Signaling Technology), phospho‐eIF2α (Ser51) (#3597, Cell Signaling Technology), eIF2α antibodies (#3524, Cell Signaling Technology), OXPHOS antibody (#ab110413, Abcam), slow myosin (#ab11083, Abcam), and UCP1 antibodies (#ab23841, Abcam). Protein expression was normalized to α‐Tubulin (ATUB) (#T6074, SIGMA). Horseradish peroxidase‐conjugated secondary antibodies were used: anti‐rabbit IgG (#7074, Cell Signaling Technology) or anti‐mouse IgG (#7076, Cell Signaling Technology).
6
Western Blot Analysis of Metabolic Proteins
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Proteins were isolated from tissues with lysis buffer [100 mM tris-HCl (pH 6.8), 2.0% SDS, 20% glycerol, 0.02% bromophenol blue, 5% 2-mercaptoethanol, 100 mM NaF, and 1 mM Na3VO4]. The concentration of homogenized protein lysates was determined by the Bradford method (Bio-Rad). The following antibodies were used for the detection of target proteins: UCP1 (#14670), phospho-PKA (#5661), total PKA (#4782), phospho-AMPK (#2535), and total AMPK (#2793) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against glucose transporter 4 (GLUT4; sc-1607), apelin (sc-293441), and HIF-1α (sc-13515) were purchased from Santa Cruz Biotechnology (Dallas, TX). PRDM16 (PA5-20872; Thermo Fisher Scientific, Rockford, IL) and PGC-1α (no. 66369-1-Ig; Proteintech, Rosemont, IL) were also purchased. Antibodies against β-actin and β-tubulin were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). IRDye 680 goat anti-mouse secondary antibody (1:10,000), IRDye 800CW donkey anti-rabbit secondary antibody (1:10,000), and IRDye 800CW donkey anti-goat secondary antibody (1:10,000) were purchased from LI-COR Biosciences (Lincoln, NE). The target proteins were detected using the infrared imaging system (Odyssey, LI-COR Biosciences), and intensity of band was quantified using Image Studio Lite (LI-COR Biosciences) (16 (link)).
7
Western Blot Analysis of Liver Proteins
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Fresh liver tissue homogenates were collected as described above. The cell pellets were then washed in phosphate-buffered saline (PBS) solution and lysed in RIPA buffer supplemented with protease inhibitor (Roche Diagnostics, Shanghai) on ice. The total protein concentration of the dissolved lysates was quantified using the Bradford method. A sample of 20 μg protein was electrophoresed using polyacrylamide gel (PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane using a wet transfer apparatus (Bio-Rad, Hercules, USA). The blots were then blocked with 5% skimmed milk PBST for 1 h followed by overnight binding with specific antibodies in a cold room, incubation with the secondary antibody for 1 h, and extensive washing. Finally, the reaction was detected using enhanced horseradish peroxidase (HRP) substrate (Thermo Fisher Scientific, Waltham, U.S.A.). The specific antibodies were used to detect murine NRF2 (1:500, ABCAM), HO-1 (1:500, ABCAM), NQO-1 (1:500, ABCAM), p-AMPK (1:1000, Cell Signaling Technologies), total-AMPK (1:1000, Cell Signaling Technologies), SIRT1 (1:1000, Cell Signaling Technologies), β-actin (1:5000, Cell Signaling Technologies), and the secondary rabbit or mouse IgG HRP conjugated antibody (1:3000, Thermo Fisher Scientific, USA). The expression levels of the target proteins were normalized to β-actin.
8
Western Blot Analysis of Adiponectin, AMPK, Irisin
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Western blotting was performed as described previously [16 (link),17 (link)]. Serum sample was diluted 10-fold with reducing protein sample buffer, heated at 95°C for 10 min and 10μl of the sample was analyzed by SDS-PAGE-immunoblotting assay. Antibodies against adiponectin, phospho-AMPK (Thr172), total AMPK, (Cell signaling technology, Beverly, MA) GAPDH (Bioss antibodies, Woburn, MA), irisin (Aviscera Bioscience, Santa Clara, CA), phospho-PPARγ (Ser112) and UCP1 (Abcam, Cambridge, UK) were used as primary antibodies, followed by the appropriate IgG-HRP conjugated secondary antibody. Proteins were visualized by ECL.
9
Berberine and TRAIL Synergistic Apoptosis
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RPMI tissue culture media, Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA); Berberine (catalogue number B3251, purity ≥98%) was from Sigma (St. Louis, MO), Troglitazone, TRAIL were purchased from EMD Biosciences (Gibbstown, NJ). The antibodies utilized were obtained from the following sources: poly (ADP-ribose) polymerase (PARP), caspase-3, caspase 8, caspase 9, pAMPKT172, total AMPK, AMPKα1 and α2, DR4, DR5 from Cell Signaling Technologies (Danvers, MA), FLAG from Sigma (St. Louis, MO), GAPDH from Ambion Inc. (Austin, TX); TRIzol reagent from Invitrogen, (Carlsbad, CA), RT2 PCR Profiler PCR Array PAHS-012Z (Cat # 330231) was from Qiagen (Valencia, CA).
10
Western Blot Analysis of Muscle Proteins
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Approximately 100 mg of soleus muscle tissue was taken and lysed with RIPA lysis buffer. Lysates were resolved on 12% SDS–polyacrylamide gels and were transferred to PVDF membrane (Millipore, USA). Membrane was blocked with 5% nonfat milk in TRIS buffered saline (TBS; 10 mM TRIS (pH 8.0), 150 mM NaCl) for 1 hour and probed with primary antibody of interest: phospho-AMPK (Thr172) (1:1000; Cell Signaling Technology, USA), phospho-ACC (Ser79) (1:1000; Cell Signaling Technology, USA), total AMPK (1:1000; Cell Signaling Technology, USA), total ACC (1:1000; Cell Signaling Technology, USA), and total Actin (1:1000; Santa Cruz Biotechnology, USA). Subsequently, membranes were incubated with corresponding HRP-conjugated secondary antibodies. The chemiluminescence signals were captured on photographic films using an enhanced chemiluminescence (Thermo Scientific, USA).The loading controls of phospho-ACC and phospho-AMPK in Fig. 6J were probed in parallel gels.
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