Western blot was performed, according to a previously described method (Kim et al., 2015 (link)). Protein extracts from hippocampal tissues were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein separation was performed using 10% polyacrylamide with 0.05% bis-acrylamide. Proteins were then transferred to nitrocellulose and the blots were probed with anti-PI3K mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Akt rabbit polyclonal antibody (1:1,000, Cell Signaling Technology Inc., Beverly, MA, USA), anti-GSK-3β rabbit polyclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-synapsin I mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), and anti-postsynaptic density-95 (PSD-95) mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology). Peroxidase anti-rabbit IgG (1:5,000, Vector Laboratories), and peroxidase anti-mouse IgG (1:10,000, Vector Laboratories) were used as the secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence (ECL) detection kit (Santa Cruz Biotechnology).
Peroxidase anti mouse igg
Manufactured by Vector Laboratories
Sourced in United States
Peroxidase anti-mouse IgG is a secondary antibody conjugated with the enzyme horseradish peroxidase. It is used to detect and visualize mouse primary antibodies in various immunoassays and immunohistochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase anti rabbit igg, by Vector Laboratories (6 mentions)
Rabbit polyclonal anti akt antibody, by Cell Signaling Technology (3 mentions)
Anti pi3k mouse monoclonal antibody, by Santa Cruz Biotechnology (3 mentions)
Ecl detection kit, by Santa Cruz Biotechnology (2 mentions)
Anti gsk 3β rabbit polyclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Anti p pi3k rabbit polyclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Anti p akt rabbit polyclonal antibody, by Cell Signaling Technology (2 mentions)
Mouse anti β actin monoclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence, by Santa Cruz Biotechnology (2 mentions)
Anti bdnf rabbit polyclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Ab6721, by Abcam (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
Valeric acid, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
10 protocols using peroxidase anti mouse igg
1
Western Blot Analysis of Hippocampal Proteins
2
Adipocyte Differentiation Reagents and Antibodies
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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), bovine calf serum, phosphate-buffered saline (PBS), and trypsin-EDTA were from Gibco BRL (Grand Island, NE, USA). Penicillin/streptomycin was from Thermo Scientific (Rockford, IL, USA). Propionic acid, valeric acid, 2-deoxy-D-glucose, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), insulin, and 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, MO, USA). 2-Deoxy-[3H]-glucose was obtained from PerkinElmer Life Sciences (Boston, MA, USA). Rosiglitazone was purchased from Masung & Co., Ltd (Seoul, Korea). The anti-GPR41 (H-100) antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The anti-PPARγ (D69) antibody was from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-β-actin and goat anti-rabbit antibodies were from Abfrontier (Geumcheon, Seoul, Korea). The anti-myosin heavy chain (MHC; MF 20) antibody was obtained from the Development Studies Hybridoma Bank (Iowa City, IA, USA). Horseradish peroxidase-conjugated secondary antibodies (peroxidase anti-rabbit IgG produced in goat, #PI-1000; peroxidase anti-mouse IgG produced in horse, #PI-2000) to detect the primary antibodies were purchased from Vector Laboratories Inc. (Burlingame, CA, USA). Other chemicals were of analytical grade.
3
Protein Expression Analysis in Spinal Cord
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Protein extracts from C5–C7 ventral horns of adult animals 7 days after surgery were analyzed on 10% sodium dodecylsulfate polyacrylamide gels and then transferred to 0.45 μm nitrocellulose membranes. The following primary antibodies were used: rabbit anti-cleavage Capase3 (1:1000; Cat No. 9661, Cell Signaling Technology), rat anti-MHC1 (1:500; sc-59199, Santa Cruz), rabbit anti-GAPDH (1:1,0000; Cat No. 5174, Cell Signaling Technology), anti-β-tubulin (1:10,000; rabbit, Cat No. 2146, Cell Signaling Technology); the secondary antibodies included Peroxidase anti-rabbit IgG (1:5,000, ab6721, Abcam) and peroxidase anti-mouse IgG (1:10,000; Vector Laboratories). Immunoreactivity was detected using an enhanced chemiluminescence (ECL) detection kit (1,705,061, Bio-Rad).
4
Western Blot Analysis of PI3K/Akt Signaling
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Western blot was conducted according to the previous method (Kim et al., 2010 (link)). Protein extracts from brain tissue were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein separation was performed using 10% polyacrylamide with 0.05% bis-acrylamide. Proteins were then transferred to nitrocellulose and the blots were probed with anti-PI3K mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-p-PI3K rabbit polyclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-Akt rabbit polyclonal antibody (1:1,000, Cell Signaling Technology), and anti-p-Akt rabbit polyclonal antibody (1:1,000, Cell Signaling Technology). Peroxidase anti-rabbit IgG (1:5,000, Vector Laboratories), and peroxidase anti-mouse IgG (1:1,000, Vector Laboratories) were used as the secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence (ECL) detection kit (Santa Cruz Biotechnology). Film auto-radiograms were exposed from 5 to 15 min.
5
Western Blot Analysis of BDNF in Spinal Cord
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Protein extracts from spinal cord tissue were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein separation was performed using a 10% polyacrylamide with 0.05% bis-acrylamide. Proteins were then transferred to nitrocellulose and the blots were probed with anti-BDNF rabbit polyclonal antibody (1:1,000, Santa Cruz Biotechnology) and anti-β-actin mouse monoclonal antibody (1:3,000, Santa Cruz Biotechnology). Peroxidase anti-rabbit IgG (1:5,000, Vector Laboratories, Burlingame, CA, USA), and peroxidase anti-mouse IgG (1:5,000, Vector Laboratories) were used as a secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence (Santa Cruz Biotechnology). Film autoradiograms were exposed from 5 to 15 min.
6
Spinal Protein Expression Analysis
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After 12-week exercise, animals (n = 3 in each group) were killed and proteins were extracted from C5 to C7 spinal segments, which were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose and the blots were probed with anti-myelin basic protein (anti-MBP) rabbit polyclonal antibody (1:1,000, ab40390, Abcam), anti-TH rabbit polyclonal antibody (1:1,000, AB152, Millipore), anti-synaptophysin mouse polyclonal antibody (SY38; 1:500, ab8049, Abcam), anti-postsynaptic density-95 (anti-PSD-95) rabbit polyclonal antibody (1:500, 516900, Invitrogen), anti-insulin like growth factor-1 (anti-IGF-1) rabbit polyclonal antibody (1:2,000, ab9572, Abcam), anti-glial cell line-derived neurotrophic factor (anti-GDNF) rabbit polyclonal antibody (1:500, ab18956, Abcam), anti-BDNF (1:500, ab108319, Abcam), anti-β actin rabbit polyclonal antibody (1:5,000; ab8227, Abcam), anti-β tubulin rabbit polyclonal antibody (1:5,000; ab6046, Abcam). Peroxidase anti-rabbit IgG (1:5,000, ab6721, Abcam) and peroxidase anti-mouse IgG (1:10,000, Vector Laboratories) were used as secondary antibodies. Immunoreactivity was detected using an enhanced chemiluminescence (ECL) detection kit (1705061, Bio-Rad).
7
Quantifying BDNF and Synapsin-I in Spinal Cord
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Western blot for BDNF and synapsin-I was performed according to the previously described method (Jung et al., 2016 (link)). The spinal cord tissues covering approximately 1 cm of the rostral and caudal spinal cord at the injury area were prepared and washed with ice-cold PBS and sonicated in 400–600 mL of Triton lysis buffer. Protein separation was performed using a 10% polyacrylamide with 0.05% bis-acrylamide. Proteins were then transferred to nitrocellulose and the blots were probed with anti-BDNF rabbit polyclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-synapsin-I rabbit monoclonal antibody (1:1,000, Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin mouse monoclonal antibody (1:3,000, Santa Cruz Biotechnology). Peroxidase anti-rabbit IgG (1:5,000; 1:5,000 Vector Laboratories, Burlingame, CA, USA), and peroxidase anti-mouse IgG (1:5,000, Vector Laboratories) were used as the secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence (Santa Cruz Biotechnology).
8
Quantitative Proteomics of Motor Neuron Markers
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Protein extracts from E13.5 mouse cervical segments, C5–C7 ventral horns of adult animals 3 days after surgery, 5-DIV cultured human motor neuron explants and proteins eluted from glutathione-agarose beads were analysed on 10% SDS polyacrylamide gels and then transferred to 0.45-μm nitrocellulose membranes. The following primary antibodies were used: anti-β-tubulin (Tuj1) rabbit polyclonal antibody (1:1000; ab18207, Abcam), anti-GAPDH mouse polyclonal antibody (1:1000; ab8245, Abcam), anti-JNK rabbit polyclonal antibody (1:1000; 9252, CST), anti-c-Jun rabbit polyclonal antibody (1:1000; 3270S, CST), anti-EB3 rat polyclonal antibody (1:1000; ab53360, Abcam), anti-GSK-3β polyclonal antibody (1:1000; 9315, CST), anti-Rac1 mouse polyclonal antibody (1:1000; 610650, BD Pharmingen), anti-CDC42 rabbit polyclonal antibody (1:1000; 11A11, CST), goat anti-human CELSR2 (1:1000, AF6379, R&D systems); the secondary antibodies include peroxidase anti-rabbit IgG (1:5000, ab6721, Abcam) and peroxidase anti-mouse IgG (1:10 000; Vector Laboratories). Immunoreactivity was detected using an enhanced chemiluminescence detection kit (1705061, Bio-Rad).
9
Western Blot Analysis of PI3K/Akt Pathway
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Western blot analysis was performed according to the previous method (Cho et al., 2017 (link); Kim et al., 2010 (link)). Protein separation was performed using 10% polyacrylamide with 0.05% bis-acrylamide. Proteins were then transferred to nitrocellulose and the blots were probed with anti-PI3K mouse monoclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-PI3K rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology), anti-Akt rabbit polyclonal antibody (1:1,000; Cell Signaling Technology Inc., Beverly, Massachusetts, USA), anti-p-Akt rabbit polyclonal antibody (1:1,000; Cell Signaling Technology), anti-GSK-3β rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology), and anti-p-GSK-3β rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology). Peroxidase anti-rabbit IgG (1:5,000; Vector Laboratories) and peroxidase anti-mouse IgG (1:10,000; Vector Laboratories) were used as the secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence (ECL) detection kit (Santa Cruz Biotechnology).
10
Mitochondrial Function and Oxidative Stress Assay
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Samples of rat liver were homogenized as previously described [38 (link)]. Liver lysates containing 30 µg protein were loaded in each lane and were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membrane. Membranes were blocked and after probed with the following antibodies: anti-PGC1α (Millipore, Burlington, MA, USA), anti-NRF1 (Abcam, Cambridge, UK), anti-TFAM (Abcam), anti-TOM20 (Cell signaling), anti-GPX4 (Abcam), anti-PRDX3 (Abcam), anti-SOD2/MnSOD (Abcam), anti-CAT (Abcam), anti-DRP1 (Abcam), anti-OPA1 (Abcam), anti-MNF2 (Abcam), anti-PAMPK Thr172 (Cell signaling, Danvers, MA, USA), anti-AMPK (Cell signaling), anti-PULK Ser555 (Cell signaling), anti-ULK1 (Cell signaling), anti-AMBRA1 (Cell signaling), anti-PINK1 (Cell signaling ) anti-PARKIN (Cell signaling), anti-LC3B (Novus biologicals), anti-POLγ (Novus biologicals, Bio-Techne SRL, Milano, Italy), anti-Total OXPHOS complexes cocktail (Abcam), and anti-βACTIN (Sigma Aldrich, St. Louis, MO, USA). As secondary antibodies, peroxidase anti-rabbit IgG (Vector Laboratories Burlingame, CA, USA) and peroxidase anti-mouse IgG (Vector Laboratories) were used. Horseradish peroxidase-conjugated secondary antibodies were detected with enhanced chemiluminescence.
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