Rabbit bdnf antibody

BDNF and TrkB expressions were determined by Western blot analysis (Kim et al., 2006 (link); Kim et al., 2015 (link)). The hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50-mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (pH, 7.5), 150-mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1-mM ethyleneglycol- bis-(b-aminoethylether)-N,N,N′,N′-tetraacetic acid, 1.5-mM MgCl₂·6H₂O, 1-mM sodium orthovanadate, and 100-mM sodium flouride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). The protein (30 μg) was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. Mouse beta-actin antibody (1:1,000; Santa Cruz Biotechnology), rabbit BDNF antibody (1:500; Santa Cruz Biotechnology), rabbit TrkB antibody (1:1,000; Santa Cruz Biotechnology), were used as the primary antibodies. Horseradish peroxidase conjugated antirabbit antibody for BDNF and TrkB (1:3,000; Vector Laboratories) and horseradish peroxidase-conjugated antimouse antibody for beta-actin (1:2,000; Vector Laboratories) were used as the secondary antibodies. Band detection was performed using the enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).

BDNF expression was determined by western blot as the previously described method (Kim et al., 2010 (link)). The hippocampal tissues were collected, and then are immediately frozen at −70°C. The hippocampal tissues was 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% SDS, 1 mM PMSF, 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. Mouse β actin antibody (1:3,000; Santa Cruz Biotechnology), rabbit BDNF antibody (1:1,000; Santa Cruz Biotechnology).
Horseradish peroxidase-conjugated anti-rabbit antibody for BDNF was used as the secondary antibody. Experiment was performed in normal lab conditions and at room temperature except for transferred membrane. Membrane transfer was performed at 4°C with the cold pack and prechilled buffer. Band detection was performed using the enhanced chemiluminescence (ECL) detection kit (Santa Cruz Biotechnology). To compare relative expression of proteins, detected bands were calculated densitometrically using Molecular Analyst™ version1.4.1 (Bio-Rad).

Expressions of BDNF and TrkB were assessed by western blot analysis, according to the previously described method (Kim et al., 2010 (link); Park et al., 2014 ). The hippocampal tissues were dissected and collected, and then were immediately frozen at −70°C. The right hemisphere were homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl₂·6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Hercules, CA, USA). Protein samples (30 μg) were separated on sodium dodecyl sulfate-polyacryl-amide gel and transferred onto a nitrocellulose membrane. The membranes were incubated with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 and then incubated overnight at 4°C with the following primary antibodies: mouse β-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit BDNF antibody (1:500; Santa Cruz Biotechnology), rabbit TrkB antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Subsequently, membranes were incubated for 1 h with attempt secondary antibodies (1:2,000; Vector Laboratories), and ban detection was performed using the enhanced chemiluminescence (ECL) detection kit (Santa Cruz Biotechnology).

Western blotting for BDNF and TrkB expressions was performed, according to the previously described method (Kim et al., 2014 (link)). The hippocampal tissues were collected, and then were immediately frozen at −70°C. The hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl₂ 6H₂O, 1 mM sodium orthovanadate, and 100 mM sodium flouride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Protein (30 μg) was separated on SDS-polyacrylamide gels and transferred onto a nitro-cellulose membrane. Mouse actin antibody (1:1,000; Santa Cruz Biotechnology), rabbit BDNF antibody (1:1,000; Santa Cruz Biotechnology) and rabbit TrkB antibody (1:1,000; Santa Cruz Biotechology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-rabbit antibody for BDNF (1:2,000; Vector Laboratories) and TrkB (1:3,000; Vector Laboratories) were used as the secondary antibodies. Band detection was performed using as enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech GmbH, Freiburg, Germany).

Western blot analysis for BDNF, TrkB, COX-2, iNOS were performed according to the previous method (Baek et al., 2016 (link); Park et al., 2017 (link)). Sciatic nerve tissue samples were homogenized in the lysis buffer and 200 μg the samples were centrifuged at 14,000 rpm at 20 min and supernatant collected for Western blot. Protein concentration in the sample was assayed using Bradford reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA). Protein of 35 μg was separated sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membrane (Whatman GmbH, Dassel, Germany). Rabbit BDNF antibody (1:1,000; Santa Cruz Biotechnology), rabbit TrkB antibody (1:1,000; Santa Cruz Biotechnology), goat COX-2 antibody (1:1,000; Santa Cruz Biotechnology), and rabbit iNOS antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-rabbit antibody (1:2,000; Vector Laboratories) for BDNF, TrkB, iNOS, and anti-goat antibody (1:2,000; Santa Cruz Biotechnology) for COX-2 were used as the secondary antibodies. Band detection was performed using an enhanced chemiluminescence detection system (Santa Cruz Biotechnology).

Western blotting was conducted according to previously published methodology [8 (link),16 (link)]. The hippocampal tissues were homogenized on ice and lysed in a lysis buffer. Protein (30 μg) was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. Mouse beta-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse GAP43 antibody (1:1,000; Santa Cruz Biotechnology), rabbit BDNF antibody (1:500; Santa Cruz Biotechnology), rabbit TrkB antibody (1:1,000; Santa Cruz Biotechnology), rabbit PSD95 antibody (1:1,000; Santa Cruz Biotechnology), rabbit Egr-1 antibody (1:1,000; Santa Cruz Biotechnology), and goat pCREB antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-rabbit antibody for BDNF, TrkB, PSD95, and Egr-1 (1:3,000; Vector Laboratories, Burlingame, CA, USA), horseradish peroxidase-conjugated anti-mouse antibody for beta-actin and GAP43 (1:2,000; Vector Laboratories), and horseradish peroxidase-conjugated anti-goat antibody for p-CREB (1:5,000; Santa Cruz Biotechnology) were used as the secondary antibodies. Band detection was conducted using an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).

Western blot analysis was conducted for the determination of BDNF and TrkB expressions, as the previously described method (Kim et al., 2010 (link)). Hippocampal tissues were collected and then immediately frozen at −70°C. Hippocampal tissues were homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl₂·6H₂O, 1 mM sodium orthovanadate, and 100 mM sodiumfluoride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Hercules, CA, USA). Protein (30 μg) was separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. Mouse beta-actin antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit BDNF antibody, and rabbit TrkB antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-rabbit antibody (1:3,000; Vector Laboratories, Burlingame, CA, USA) for BDNF and TrkB were used as the secondary antibodies. Experiments were performed under normal laboratory conditions and at room temperature, except for the transfer to membranes. The transfer was performed at 4°C with a cold pack and pre-chilled buffer. Band detection was performed using enhanced chemiluminescence (ECL) detection kits (Santa Cruz Biotechnology).