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.
Enhanced chemilluminescence
Manufactured by Thermo Fisher Scientific
Enhanced chemiluminescence is a sensitive, quantitative technique used for detecting and measuring proteins in Western blotting analysis. It involves the generation of light through a chemical reaction, which is then detected and quantified using specialized imaging equipment.
Automatically generated - may contain errors
2 protocols using enhanced chemilluminescence
1
Leptin-induced Akt Phosphorylation in Hippocampus
2
Leptin-induced Akt Phosphorylation in Hippocampus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!