Cell Counting Kit-8 (CCK-8), Bicinchoninic acid (BCA) Protein Assay Kits, Annexin V-FITC Apoptosis Detection Kits, and Cell Cycle Analysis Kits were all purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). Protein Extraction Kits were obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). Diaminobenzidine (DAB) substrate kits were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) kits were provided by Roche Diagnostics (Germany). 4′,6′-Diamidino-2-phenylindole (DAPI), tris (hydroxymethyl) aminomethane (Tris), and sodium dodecyl sulphate (SDS) were purchased from Sigma (St. Louis, MO, U.S.A.). Anti-LOX1, anti-caspase-9, anti-p65, anti-p-IKKβ, anti-IKKβ, anti-p-IkBα, anti-IkBα, anti-GAPDH, anti-β-tubulin, and anti-Lamin B primary antibodies, and horseradish peroxidase-conjugated antibody were obtained from Abcam (Cambridge, U.K.). Plasmids harboring the wild-type caspase-9 response element (WT-luciferase-caspase-9) and the corresponding mutant (MUT-luciferase-caspase-9) were purchased from Vipotion Biotechnology (Guangzhou, China). Pierce Agarose Chip Kits were obtained from Thermo Fisher Scientific (Waltham, MA, U.S.A.).
Horseradish peroxidase conjugated antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Horseradish peroxidase-conjugated antibodies are a type of labeled antibody used in various immunoassay techniques. They consist of an antibody molecule covalently linked to the enzyme horseradish peroxidase. This enzyme catalyzes a colorimetric reaction, allowing for the detection and quantification of the target antigen in a sample.
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Lab products found in correlation
Pb0053, by Boster Bio (2 mentions)
Ba0406, by Boster Bio (2 mentions)
Ba0615, by Boster Bio (2 mentions)
Enhanced chemiluminescence kit, by Santa Cruz Biotechnology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Anti trka, by Santa Cruz Biotechnology (2 mentions)
Anti ptrka, by Santa Cruz Biotechnology (2 mentions)
Anti akt1 2 3, by Santa Cruz Biotechnology (2 mentions)
Dulbecco s modified eagle s medium, by Cytiva (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Poly d lysine, by BD (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Cell cycle analysis kit, by Beyotime (1 mentions)
Sodium dodecyl sulphate (sds), by Merck Group (1 mentions)
Anti β tubulin, by Abcam (1 mentions)
Anti caspase 9, by Abcam (1 mentions)
Anti ikbα, by Abcam (1 mentions)
Anti p ikbα, by Abcam (1 mentions)
Anti ikkβ, by Abcam (1 mentions)
11 protocols using horseradish peroxidase conjugated antibody
1
Assessing Cellular Behavior and Signaling
2
Western Blot Detection of MAPK and NF-κB Signaling
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Detections of phospho-p44/42 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, and NF-κB p65 were performed using western blot. After PA treatment, cells lysed in buffer containing 1% Triton X-100 (Sigma-Aldrich) and Protease/Phosphotase Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). Protein concentrations assessed with Quick Start Bradford Protein Assay kit following manufacturer's protocol. Lysates loaded with Laemmli Sample Buffer onto Any kD Mini-PROTEAN-TGX pre-cast polyacrylamide gel (Bio-Rad) and separated by electrophoresis. Gels transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) and detected with primary antibodies for MAPK (Cell Signaling) or NF-κB (Sigma-Aldrich) pathways, secondary horse radish peroxidase-conjugated antibody (Abcam, Cambridge, England), and chemiluminescent substrate (Thermo Fisher Scientific) before visualization with ChemiDoc MP System (Bio-Rad).
3
Quantifying Adhesion Molecule Expression in HUVECs
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Adhesion molecules expression was measured by immunocytochemistry. Briefly, HUVECs were plated in 96-well plates. Confluent HUVECs were serum starved for 6 h and treated with 15-oxoETE (0, 0.5, 1, 2, 4, 8 μmol/L) for 6 h at 37 °C, TNF-α was served as positive control. Then HUVECs were incubated with Rabbit Anti-ICAM-1 (PB0053, Boster) or Rabbit Anti -VCAM-1 (BA0406, Boster) or Rabbit Anti -E-selectin (BA0615, Boster) (1:200; Boster, China) in First Antibody Dilution Buffer for 2 h 37 °C. Omission of primary antibody was conducted in Negative controls. And then cells were incubated with a horseradish peroxidase-conjugated antibody (1:1000, Abcam, USA) for 1.5 h at 37 °C. Quantification was performed by measuring the absorbance at 450 nm by a TMB peroxidase EIA substrate kit (Bio-Med Innovation, China) [22 (link)].
4
Protein Expression Analysis in A2780 Cells
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Protein expression in A2780 and A2780cisR cells was analyzed as described previously [32] (link). The proteins were extracted in denaturation buffer (Tris pH 6.8, 20% SDS, glycerol and mercaptoethanol) and separated on 13% acrylamide gel. The primary antibodies were purchased as follow: GCLM, GSR, GST3/GSTP, β-tub (Rabbit monoclonal Abcam, Cambridge, Massachusetts, USA). β Tubulin was used as loading control.
Primary antibodies were detected using a horseradish peroxidase-conjugated antibody and enhanced chemiluminescence (Abcam, Cambridge, Massachusetts, USA). The films were scanned, and band intensity was quantified using densitometry software (ImageJ, National Institutes of Health, Bethesda, Maryland, USA).
Primary antibodies were detected using a horseradish peroxidase-conjugated antibody and enhanced chemiluminescence (Abcam, Cambridge, Massachusetts, USA). The films were scanned, and band intensity was quantified using densitometry software (ImageJ, National Institutes of Health, Bethesda, Maryland, USA).
5
TMAO Regulates Adhesion Molecules in HUVECs
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Adhesion molecules expression was measured by immunocytochemistry. Briefly, HUVECs were plated in 96-well plates. Confluent HUVECs were serum starved for 6 h and treated with TMAO (0, 10, 50, and 100 μmol/l) for 6 h at 37°C, TNF-α was served as the positive control. Then, HUVECs were incubated with rabbit anti-ICAM-1 (PB0053, Boster) or rabbit anti-VCAM-1 (BA0406, Boster) or rabbit anti-E-selectin (BA0615, Boster) (1:200; Boster, China) in First Antibody Dilution Buffer for 2 h at 37°C. Omission of primary antibody was conducted in negative controls. And then the cells were incubated with a horseradish peroxidase-conjugated antibody (1:1000, Abcam, U.S.A.) for 1.5 h at 37°C. Quantification was performed by measuring the absorbance at 450 nm by a TMB peroxidase EIA substrate kit (Bio-Med Innovation, China) [25 (link)]. PKC inhibitor, staurosporine (2.5 nmol/l), was used at the beginning of the treatment.
6
Immunohistochemical Analysis of NLRP1 and NLRP3 in Gastric Cancer
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We collected 20 cases of primary GC and corresponding adjacent normal tissue samples and performed immunohistochemical staining. Briefly, GC sections were incubated with monoclonal antibodies against NLRP1/NLRP3 (all from Abcam Company, Cambridge, UK) at a 1:100 dilution overnight at 4°C. The sections were conjugated with horseradish peroxidase-conjugated antibodies (Abcam) at a 1:500 dilution for 2 h at room temperature. Then 3,3-diaminobenzidine was added. The slides were mounted with Vectashield mounting medium. Light microscopy (Olympus) was used to observe the samples. Immunohistochemical scores were determined independently by two pathologists as previously described [29 (link)]. The experimental samples were collected from September to December 2021 in the Oncology Department of Changshu Second People's Hospital (Supplementary Table 1 ).
7
Western Blot Protocol for Reelin and Actin
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All Western blots (WBs) were performed with the WB-system BIORAD Mini-PROTEAN Tetra System. The protocol was adapted from Trotter (Trotter et al. 2014 (link)). In brief, the tissue was homogenized in M-PER and HALT (Thermo Fisher), supplemented with 3× Laemmli buffer and incubated at 95 °C for 10 min. To obtain dentate gyrus samples, freshly dissected hippocampi were cut into 500 μm sections using a McIllwan tissue chopper and the dentate gyrus was subsequently excised from five slices. Acrylamide gels with 8% and 15% running gels stacked on each other were used to separate Reelin and Actin on the same gel. The membrane was cut at the level of the 100 kDa marker band, blocked with 5% milk powder in tris-buffered saline with 0.1% Tween20 (TBST20) for 1 h at RT before incubation with mouse anti-Reelin antibody (Merck Millipore MAB5364) and rabbit anti-Actin antibody (Sigma-Aldrich A-2066), each diluted 1:1000 in 5% milk powder in TBST20 overnight. Horseradish peroxidase-conjugated antibodies from Abcam were used as secondary antibodies and the signals were detected with SuperSignal WestPico Chemiluminescent Substrate Kit (Fisher Scientific). Signal intensity was analyzed using the Fiji package of the ImageJ software (Schindelin et al. 2012 (link); Schneider et al. 2012 (link)).
8
Synthesis and Evaluation of NGF-Based Dipeptides
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The dimeric dipeptides GK-6 and GK-2 were synthesized on the base of murine NGF at the Zakusov Institute of Pharmacology (Moscow, Russia).
Inhibitors of PI3K (LY294002) and MAPK (PD98059) were purchased from Tocris Bioscience (Bristol, UK). The tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT),was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals. Inc. (Costa Mesa, CA, USA). Poly-D-lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Biorad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-pAKT1/2/3, anti-ERK1/2, anti-pERK1/2 antibodies and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase conjugated antibodies were purchased from Abcam (Cambridge, MA, USA).
Inhibitors of PI3K (LY294002) and MAPK (PD98059) were purchased from Tocris Bioscience (Bristol, UK). The tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT),was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals. Inc. (Costa Mesa, CA, USA). Poly-D-lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Biorad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-pAKT1/2/3, anti-ERK1/2, anti-pERK1/2 antibodies and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase conjugated antibodies were purchased from Abcam (Cambridge, MA, USA).
9
Synthesis and Characterization of Dimeric Dipeptides
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The dimeric dipeptides GSB-106 ((bis-(N-monosuccinyl-l -seryl-l -lysine)hexamethylenediamide (Tm =143°C–145°C, [α] 20 (link) D =−24.7° (c=0.4%; dimethylformamide)) and GSB-214((bis-(N-monosuccinyl-l -methionyl-l -serine) heptamethylenediamide (Tm =160°C–162°C, [α] 20 (link) D =−21.75° (c=0.4%; MeOH)) were synthesized at the Zakusov Institute of Pharmacology (Moscow, Russia), as described previously.13 (link)
2,3,5-triphenyltetrazolium chloride (TTC) and Nembutal were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA). Poly-d -lysine was purchased from BD Biosciences (San Jose, CA, USA). DC protein assay was purchased from Bio-Rad (Hercules, CA, USA). Anti-TrkA, anti-pTrkA, anti-AKT1/2/3, anti-phospho-AKT1s473/2s472/3s474 anti-ERK1/2, anti-phospho ERK1/2Y204 antibodies, and enhanced chemiluminescence kits were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-β-actin antibodies and horseradish peroxidase-conjugated antibodies were purchased from Abcam (Cambridge, MA, USA). Formalin was purchased from Ecros (Saint Petersburg, Russia). GSB-106 and GSB-214 were dissolved in water. Then solvents were diluted in culture media in equivalent amounts.
2,3,5-triphenyltetrazolium chloride (TTC) and Nembutal were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium was purchased from HyClone Laboratories (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Langley, OK, USA). Glutamine was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA). Poly-
10
Western Blot Analysis of Cellular Proteins
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The cells were treated as above and lysed in lysis buffer. Protein concentrations were determined using a commercial kit (Bradford Protein Assay, Biorad, UK) according to the manufacturer's specifications. Proteins were separated on 12% polyacrylamide gels and transferred to polyvinylidenefluoride membranes. These membranes were incubated for 1 h at room temperature in Tris-buffered saline containing 0.5% non-fat milk powder, and 0.1 % Tween-20. An antibody against vimentin (Abcam, Cambridge, UK), an antibody against p53 (Abcam, Cambridge, UK), and an antibody against bcl-2 (Abcam, Cambridge, UK) were used. An antibody against GAPDH (diluted 1:1,000; Abcam, Cambridge, UK) was used as the positive control. After incubation with the primary antibody overnight at 4 °C, the membranes were washed three times in TBS. After incubation with the secondary horseradish peroxidase-conjugated antibodies (1:1,000; Abcam, Cambridge, UK) for 1 h at room temperature, detection was performed using an enhanced chemiluminescence assay kit (GE Healthcare, UK). Digital images were captured and density measurements were made using commercial software (Quanity One, Biorad).
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