HepG2 and C2C12 cells were purchased from ATCC (Manassas, VA). DMEM, Penicillin–Streptomycin (PS), fetal bovine serum (FBS) and horse serum (HS) were from Invitrogen (Grand Island, NY). D-(+)-Glucose solution, 45%, insulin, insulin-like growth factor-1 (IGF-1), and Akt inhibitor VIII were purchased from Sigma–Aldrich (St. Louis, MO). Rapamycin was purchased from LC Laboratories (Woburn, MA), and wortmannin was from Adipogen (San Diego, CA). SAMS peptide was purchased from Abcam (Cambridge, MA) and P32 was from Perkin–Elmer (Boston, MA).
Sams peptide
Manufactured by Abcam
Sourced in United Kingdom
SAMS peptide is a synthetic peptide that can be used for various research applications. It has a defined sequence and is suitable for use in a range of experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Wortmannin, by Adipogen (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
D glucose solution, by Merck Group (1 mentions)
Insulin like growth factor 1 igf 1, by Merck Group (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Akt inhibitor 8, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
C2c12 cell, by American Type Culture Collection (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Insulin, by Merck Group (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
Gamma 32p atp, by PerkinElmer (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
Nucview 488, by Biotium (1 mentions)
Cptio, by Dojindo Laboratories (1 mentions)
Adp glo kinase assay kit, by Promega (1 mentions)
Draq7, by Biostatus (1 mentions)
4 protocols using sams peptide
1
HepG2 and C2C12 Cellular Assays
2
AMPK Activity Measurement Protocol
3
AMPK Activity Assay Protocol
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Cells were washed twice with 1× PBS and lysed in 50 mM Tris, 250 mM mannitol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1% triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate plus protease inhibitor tablet (Roche, 1 tablet/10 ml). Protein content was normalized by BCA assay. An equal amount of protein was added to AMPK activity assay buffer containing 62.5 mM HEPES, 62.5 mM NaCl, 1.25 mM EDTA, 1.25 mM EGTA, 1 mM DTT, 5 mM MgCl2, 1 mM adenosine monophosphate, 2.5 mM adenosine triphosphate, 50 µCi ATP Gamma 32 P (10 mCi/ml, Perkin Elmer), 6.25 mM sodium pyrophosphate plus protease inhibitor tablet. Samples were incubated with and without 1 mM SAMS peptide (Abcam) for 10 min at 30 °C. Reactions were spotted on P81 Whatman paper square (EMD Millipore) and air dried. Squares were washed three times for five minutes with 1% phosphoric acid, once for five minutes in water, and once in acetone for five minutes. Squares were then air dried, added to scintillation fluid and counted as described above. Radioactive counts in samples without SAMS peptide was considered background.
4
AMPK Activity Measurement using ADP-Glo Assay
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Caspase-3 activity was determined using the NucView488 and RedDot2 apoptosis and necrosis kit (Biotium), according to the manufacturer's instructions. Dead cells were stained with RedDot2 or DRAQ7 (BioStatus, Shepshed, UK). HeLa cells were incubated with the vehicle, 0.2 mM cPTIO (Dojindo), or 4 mM L-NAME (Nacalai Tesque) for 1 h before L-c414 was added. The data were analyzed by using the Accuri C6.
In vitro kinase assay For the assay, 0.5 ng/ml full-length recombinant human AMPKa1b1g1 (SignalChem, Richmond, BC, Canada) was incubated for 60 min with 0.2 mg/ml of SAMS peptide (Abcam, Cambridge, UK) and 150 mM ATP in the kinase assay buffer [2.5 mM MOPS (pH 7.2), 1.25 mM b-glycerol-phosphate, 2.5 mM MgCl 2 , 0.5 mM EGTA, 0.2 mM EDTA, 0.25 mM DTT, 0.01 % (w/v) Brij-35, 0.25 % (w/v) bovine serum albumin (BSA), and 0.125 % (w/v) Tween 80] at 25 C. ATP hydrolysis resulting from the kinase reactions was detected using the ADP-Glo kinase assay kit (Promega).
In vitro kinase assay For the assay, 0.5 ng/ml full-length recombinant human AMPKa1b1g1 (SignalChem, Richmond, BC, Canada) was incubated for 60 min with 0.2 mg/ml of SAMS peptide (Abcam, Cambridge, UK) and 150 mM ATP in the kinase assay buffer [2.5 mM MOPS (pH 7.2), 1.25 mM b-glycerol-phosphate, 2.5 mM MgCl 2 , 0.5 mM EGTA, 0.2 mM EDTA, 0.25 mM DTT, 0.01 % (w/v) Brij-35, 0.25 % (w/v) bovine serum albumin (BSA), and 0.125 % (w/v) Tween 80] at 25 C. ATP hydrolysis resulting from the kinase reactions was detected using the ADP-Glo kinase assay kit (Promega).
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