Dorsal spinal cord tissues at the L5 and L6 levels were removed, dissected, and homogenized in 300 μl RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, and 1 mM NaF in the presence of the protease inhibitor cocktail. Samples were then put on ice for 30 min with shaking. Lysates were centrifuged at 13,000 g for 30 min at 4°C and the supernatant was collected. The protein concentration was quantified using a DC protein assay kit (Bio-Rad, Hercules, CA). Thirty μg of total proteins of each sample was loaded and separated on 4–12% Bis-Tris SDS-PAGE (Invitrogen). The resolved proteins were transferred to nitrocellulose membranes. The membranes were treated with 5% bovine serum albumin in Tris buffer containing Tween 20 for 2 h and then incubated with a rabbit anti-nNOS antibody (Cat. #07-571-I, EMD Millipore) overnight at 4°C. The membrane was washed three times and then incubated with horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. The protein band was revealed with an ECL Plus detection kit (ThermoFisher, Rockfort, IL), and the protein band intensity was quantified by using the ImageJ software program. The amounts of proteins were normalized by GAPDH (Cell Signaling Technology), which was used as a protein loading control.
Horseradish peroxidase conjugated goat anti rabbit secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States, Cameroon
Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody is a reagent used in various immunoassay and immunohistochemistry techniques. It consists of a goat-derived antibody that binds to rabbit primary antibodies, with the horseradish peroxidase enzyme conjugated to it. This enzyme can be used to catalyze a color-producing reaction, allowing for the detection and visualization of target proteins or antigens.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Wet transfer system, by Bio-Rad (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Rabbit anti ptprn2 antibodies, by Merck Group (2 mentions)
Rabbit anti gfp, by Abcam (2 mentions)
Rabbit anti akt, by Abcam (2 mentions)
Rabbit anti pi3k, by Abcam (2 mentions)
Rabbit anti mtor, by Abcam (2 mentions)
Rabbit anti rab35, by Abcam (2 mentions)
Bis tris sds page, by Thermo Fisher Scientific (1 mentions)
Ecl plus detection kit, by Thermo Fisher Scientific (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Proteinase inhibitor cocktail p8340, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
23 protocols using horseradish peroxidase conjugated goat anti rabbit secondary antibody
1
Western Blot Analysis of nNOS in Spinal Cord
2
Western Blot Analysis of KMO Levels
3
Western Blot Analysis of Fly Heads
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10 adult fly heads/sample were prepared in sample buffer using standard methods. SDS-PAGE was performed using the Novex NuPAGE SDS-PAGE system with 4%-12% Bis-Tris gels run at 125 V for 10 minutes and 150 V for 2.5 hours. Transfer to PVDF membrane (Bio-Rad, Hercules, CA) was performed using a Trans-Blot-SDSemi-Dry Transfer Cell (Bio-Rad, Hercules, CA). Blocking was performed in 5% BSA for GFP blots or 5% milk for actin blots in 1X PBS with 0.1% Tween 20. Primary antibodies were obtained from the DSHB, mouse anti-actin (JLA20) 1:1000, or from Torrey Pines Biolabs, rabbit anti-GFP 1:2000. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at 1:5000 for actin blots. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at 1:5000 for GFP blots. All antibodies were diluted in blocking buffer. Blots were developed with Super-Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA) and imaged with Amersham Hyperfilm ECL film (GE Healthcare Limited, Buckinghamshire, UK). Band intensity was quantified using ImageJ.
4
Western Blot Analysis of APP Expression
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Cells were lysed in RIPA lysis solution plus containing 2 μM PMSF on ice. Cell lysate was collected using a cell scraper and centrifuged at 13,780 × g for 5 minutes. The isolated proteins were quantified using a bicinchoninic acid assay (Thermo-Fisher Scientific) and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, with up to 40 μg protein in each lane. Proteins were then transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA) and then incubated with rabbit anti-APP primary antibody (diluted 1:20,000, cat# ab32136; Abcam, Cambridge, MA, USA) or rabbit anti-GAPDH primary antibody (diluted 1:4000, Cat# 25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (diluted 1:5000, Cat# 111-035-003; Jackson, West Grove, PA, USA) at room temperature for 45 minutes. The bands were visualized using western blot enhanced chemiluminescence substrate (Bio-Rad, Hercules, CA, USA). Relative expression among groups was quantified by ImageJ software. GAPDH was used as a reference control.
5
Quantitative ELISA-based Protein Expression
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The ELISA procedure was adapted from Sung et al. (73 (link)). HEK293F cells in 96-well poly-D-Lysine–coated plates were transfected with 50 ng per well Ca2+ assay constructs, and fixed ∼40 h to 45 h later. HTLA cells transfected with 50 ng per well Tango constructs were changed to DMEM+1% dialyzed FBS media ∼24 h later and fixed 1 d to 2 d after that. Cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, then washed with PBS and blocked in PBSA (PBS with 1% bovine serum albumin) for 15 min, followed by incubation with FLAG antibody (rabbit monoclonal, Cell Signaling #14793) diluted 1:1,000 in PBSA for 1 h. Cells were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch) 1:1,000 (0.8 μg/mL antibody) in PBSA for 30 min. Cells were washed again, 50 μL of SuperSignal Pico substrate (Thermo) were added to each well, and luminescence (all wavelengths) was read immediately. ELISAs were performed with five or six technical replicates for each mutant, along with WT and EV controls on the same plate. Replicates were averaged, and relative surface expression values were calculated by subtracting the EV value and then dividing by WT from the same plate. In some cases, negative values were obtained—these were set at zero.
6
Western Blot Analysis of Signaling Proteins
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Western blot was performed using fresh or frozen tissue, as previously described [11 (link)]. The following antibodies were used; phospho-Thr172-AMPKα (Cell Signaling Technology Cat# 2535S RRID:AB_331250), AMPKα (Cell Signaling Technology Cat# 2532S RRID:AB_10694064), phospho-Ser79-ACC Cell Signaling Technology Cat# 3661S RRID:AB_330337) and phospho-Ser428-LKB1 (Cell Signaling Technology Cat# 3482S RRID:AB_2198321), nNOS (Abcam Cat# ab1376 RRID:AB_300614), phospho nNOS Ser1417, equivalent to human Ser1412 (Abcam Cat# ab90443 RRID:AB_2049208) and β-actin (Sigma-Aldrich Cat# A3853 RRID:AB_262137), followed by horseradish peroxidase-conjugated, goat anti-rabbit secondary antibody (Jackson ImmunoResearch Cat# 111-035-045 RRID:AB_2337938). Protein concentrations were determined using protein assay dye from Bio-Rad Laboratories (Hercules, CA). Densitometry of protein expression was performed using a GS800 calibrated densitometer from Bio-Rad laboratories (Hercules, CA).
7
Western Blot Analysis of Protein Modifications
8
Western Blot Protein Detection Protocol
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Proteins were resolved on a 4–15% SDS-PAGE gel (Bio-Rad) and transferred overnight onto nitrocellulose membrane (Bio-Rad). Venus-tagged proteins were detected with the rabbit monoclonal antibody ab290 (Abcam, 1:2000) and myc-tagged proteins were detected with 71D10 rabbit monoclonal antibody (Cell Signaling, 1:1000). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson Laboratories, 1:5000) was used, followed by chemiluminescence detection with SuperSignal Femto Sensitivity Substrate (Life Technologies) and imaged with a Bio-Rad Imager. Venus-tagged protein levels were also detected using mouse monoclonal antibody B34 (Covance, 1:1000) and mouse monoclonal anti-Pgk1 antibody (ab113687, Abcam, 1:5000) was used to detect Pgk1 as a loading control.
9
Western Blot Analysis of mTOR and AMPK
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Protein from neonatal LD muscle homogenate was quantified for total protein content by BCA assay (Pierce) and subjected to western blotting. Equal amounts of protein were electrophoresed and separated on 7.5% Mini‐PROTEAN TGX Precast Gels, transferred to an Immun‐Blot PVDF Membrane (Bio‐Rad, Hercules, CA) and stained with SimplyBlue SafeStain (ThermoFisher Scientific) to ensure protein transfer. The membrane was then incubated at 4°C overnight with the following the primary antibodies at a 1:1000 dilution, rabbit anti‐phospho mTOR (Ser‐2448) and rabbit anti‐phospho‐AMPKα (Thr‐172) (Cell Signaling Technology, Danvers, MA). Membranes were incubated for 1 h with horseradish peroxidase‐conjugated goat anti‐rabbit secondary antibody (Jackson Immunoresearch, West Grove, PA), and developed with SuperSignal West Pico Chemiluminescent Substrate Kit (ThermoFisher Scientific). Densitometry analysis was performed using a ChemiDoc XRS system and Image Lab Software (BioRad). Equal loading of proteins was confirmed by reprobing with anti‐AMPKα and anti‐mTOR antibodies (1:1000, Cell Signaling Technology). Optical density was normalized to a pooled treatment sample as a loading control.
10
Comparative Analysis of Canine and Human Cancer Cell Lines
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Human (OV-2008, SKOV-3, and ECC1) and canine (Payton, CTAC-3, CMT-27, and Denny) cancer cell lines were obtained from the University of Wisconsin-Madison College of Veterinary Medicine (US). Human cell lines were used as a control to validate canine cell lines. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and incubated at 37°C in a 5% carbon dioxide (CO2) atmosphere.
The primary antibodies targeting cleaved caspase 3, superoxide dismutase (SOD), phosphorylated-focal adhesion kinase (p-FAK), and β-actin were purchased from cell signaling technologies (Danvers MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies and sheep anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch (West Grove PA, USA). All other reagents were obtained from ThermoFisher (Waltham, MA, USA).
The primary antibodies targeting cleaved caspase 3, superoxide dismutase (SOD), phosphorylated-focal adhesion kinase (p-FAK), and β-actin were purchased from cell signaling technologies (Danvers MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies and sheep anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch (West Grove PA, USA). All other reagents were obtained from ThermoFisher (Waltham, MA, USA).
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