Mice were decapitated rapidly at 30 min after i.p. injection of leptin. The hippocampi were dissected out and homogenized in lysis buffer (50 mM HEPES pH 7.6, 1% triton X-100, 150 mM NaCl, 20 mM sodium pyrophosphate, 20 mM b-glycerophosphate, 10 mM NaF) containing a mixture of phosphatase inhibitors (leupeptin, aprotinin, sodium orthovanadate, phenylmethylsulfonyl fluoride, Ser/Thr phosphatase inhibitor mixture, Tyr phosphatase inhibitor mixture). A total amount of 40 μg protein was separated by SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked in a blocking buffer (0.01 M Tris-buffered saline, 0.01M Tris base, 0.9% NaCl with 1% dry milk and 0.1% Tween 20) followed by incubation with mouse anti-Akt (1:1000; Invitrogen, Carlsbad, CA) and rabbit anti-pAktThr308, pAktSer473 or Akt antibodies (1: 1000, Cell Signaling Technology Inc., Danvers, MA) diluted in a solution of 1% bovine serum albumin and 0.1% Tween-20 in Tris-buffered saline overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G was used as secondary antibodies (1:5000, Cell Signaling). Signals were detected by enhanced chemilluminescence (Thermo Scientific, Rockford, IL). Quantification of Western blotting was performed using ImageJ software with normalization to total protein levels.
Horseradish peroxidase conjugated anti mouse or anti rabbit immunoglobulin g
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G is a secondary antibody that binds to primary antibodies raised in mice or rabbits. The horseradish peroxidase enzyme conjugated to the secondary antibody can be used to detect and visualize the presence of the primary antibody in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using horseradish peroxidase conjugated anti mouse or anti rabbit immunoglobulin g
1
Leptin-induced Akt Phosphorylation in Hippocampus
2
Leptin-induced Akt Phosphorylation in Hippocampus
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Mice were decapitated rapidly at 30 min after i.p. injection of leptin. The hippocampi were dissected out and homogenized in lysis buffer (50 mM HEPES pH 7.6, 1% triton X-100, 150 mM NaCl, 20 mM sodium pyrophosphate, 20 mM b-glycerophosphate, 10 mM NaF) containing a mixture of phosphatase inhibitors (leupeptin, aprotinin, sodium orthovanadate, phenylmethylsulfonyl fluoride, Ser/Thr phosphatase inhibitor mixture, Tyr phosphatase inhibitor mixture). A total amount of 40 μg protein was separated by SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked in a blocking buffer (0.01 M Tris-buffered saline, 0.01M Tris base, 0.9% NaCl with 1% dry milk and 0.1% Tween 20) followed by incubation with mouse anti-Akt (1:1000; Invitrogen, Carlsbad, CA) and rabbit anti-pAktThr308, pAktSer473 or Akt antibodies (1: 1000, Cell Signaling Technology Inc., Danvers, MA) diluted in a solution of 1% bovine serum albumin and 0.1% Tween-20 in Tris-buffered saline overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G was used as secondary antibodies (1:5000, Cell Signaling). Signals were detected by enhanced chemilluminescence (Thermo Scientific, Rockford, IL). Quantification of Western blotting was performed using ImageJ software with normalization to total protein levels.
3
Western Blot Analysis of Porcine Coronary Endothelial Cells
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Porcine coronary artery endothelial cells were washed with ice-cold PBS and lysed with a RIPA buffer. Equal protein concentrations (10 μg/lane) were then separated on a denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel. Subsequently, separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane at 100 V for 2 h. Next, the membranes were blocked at room temperature in TBS using bovine serum albumin in 0.1% Tween 20 for 1 h. To detect proteins, membranes were incubated with the respective primary antibody: eNOS phosphorylated at Ser1127 [p-eNOS (Ser1177)] (1:1000, Cell Signaling Technology, MA, United States), Src phosphorylated at Tyr416 [p-Src (Tyr416)] (1:1000, Cell Signaling Technology, MA, United States), and β-tubulin (1:1000, Cell Signaling Technology, MA, United States) overnight at 4°C. After washing, membranes were further incubated with the appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (1:2000, Cell Signaling Technology, MA, United States) and developed using an enhanced chemiluminescence (ECL) detection kit. Finally, band densities were determined using the ImageJ software. Expression levels of these target proteins were analyzed in at least three individual experiments.
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