Western blotting for the determination of Bax, Bcl-2, BDNF, TrkB was conducted as previously described method (Park et al., 2019 (link); Song et al., 2018 (link)). The hippocampus tissues were homogenized on ice and lysed in a lysis buffer containing 50 mM Tris-HCl (pH, 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, and 100-mg/mL leupeptin. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein of 30 μg was separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane, which was incubated with mouse β-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse Bax (1:1,000; Santa Cruz Biotechnology), Bcl-2 (1:1,000; Santa Cruz Biotechnology), and rabbit BDNF (1:1,000; Santa Cruz Bio-technology), TrkB (1:1,000; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-mouse for β-actin, Bax, Bcl-2 and anti-rabbit for BDNF, TrkB were used as secondary antibodies.
Protocol detail
Hippocampal Western Blot Analysis
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Actin
Antibodies
Antibody
Assay
Bcl 2
Buffer
Colorimetric
Deoxycholic acid
Hippocampus
Horseradish peroxidase
Leupeptin
Membrane
Mouse
Nacl
Nitrocellulose
Nonidet
Phenylmethylsulfonyl fluoride
Polyacrylamide gels
Protein
Rabbit
Sodium dodecyl sulfate
Tissues
Tris
Trkb
Corresponding Organization :
Other organizations : Temple University, Kyung Hee University, Sangmyung University, Tongmyong University
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Variable analysis
independent variables
- None explicitly mentioned
- Levels of Bax, Bcl-2, BDNF, and TrkB proteins
- Hippocampus tissue samples
- Lysis buffer composition (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, and 100-mg/mL leupeptin)
- Protein quantification using Bio-Rad colorimetric protein assay kit
- Protein separation on SDS-polyacrylamide gels and transfer onto nitrocellulose membrane
- Primary antibodies: mouse β-actin (1:1,000), mouse Bax (1:1,000), mouse Bcl-2 (1:1,000), and rabbit BDNF (1:1,000), rabbit TrkB (1:1,000)
- Secondary antibodies: Horseradish peroxidase-conjugated anti-mouse for β-actin, Bax, Bcl-2, and anti-rabbit for BDNF, TrkB
- None explicitly mentioned
- None explicitly mentioned
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