The cells were lysed using sodium dodecyl sulfate buffer containing proteinase inhibitors (Roche). Equal amounts of protein (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Shanghai, China). The membranes were blocked and incubated with Antibodies against GAPDH (dilution 1:200, Cat. no. sc-47724), IL-6 (1:200, Cat. no. sc-65327), p53 (1:200, Cat. no. sc-47698) and IL-17R (1:100, Cat. no. sc-376374) overnight at 4°C. Antibodies against GAPDH, IL-6, p53 and IL-17R were purchased from Santa Cruz Biotechnology. The membranes were then incubated with horseradish peroxidase-labeled secondary antibody (1:500, Cat. no. sc-2031) (Santa Cruz Biotechnology). The protein bands were visualized using an enhanced chemiluminescence reagent.
Horseradish peroxidase labeled secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Horseradish peroxidase-labeled secondary antibody is a laboratory reagent used in immunoassays and other applications that require the detection and visualization of target proteins or molecules. It consists of a secondary antibody conjugated to the enzyme horseradish peroxidase, which can catalyze a colorimetric or chemiluminescent reaction, allowing for the specific detection of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (10 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Thrombin, by Merck Group (4 mentions)
Fibronectin, by Santa Cruz Biotechnology (4 mentions)
Ecl western blotting detection reagent, by GE Healthcare (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Proteinase inhibitor, by Roche (3 mentions)
2 mesadp, by Merck Group (3 mentions)
G box chemi gel documentation system, by Syngene (3 mentions)
Tenascin c, by Santa Cruz Biotechnology (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Amersham hybond p, by GE Healthcare (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Prostaglandin e1 pge1, by Merck Group (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Apyrase type 5, by Merck Group (2 mentions)
Collagen 1a1, by Santa Cruz Biotechnology (2 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions)
69 protocols using horseradish peroxidase labeled secondary antibody
1
Western Blot Analysis of Cellular Proteins
2
Assessing LEP-R Expression in Iliac Bone
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In the current study, the PV-9000 two-step immunohistochemical method was used to evaluate LEP-R level in slices of iliac bone tissue (22 (link)). Briefly, paraffin-embedded sections were deparaffinized and hydrated. Next, antigen retrieval was performed in 0.01 M citric acid (pH 6.0), and the sections were then incubated with a 3% H2O2 solution for 15 min at room temperature to block endogenous peroxidase activity. Subsequently, the sections were incubated with an anti-LEP-R antibody (cat. no. sc-8325; 1:150; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight in a humid environment, followed by incubation with a horseradish peroxidase-labeled secondary antibody (cat. no. PV-9000; 1:50; OriGene Technologies, Inc., Beijing, China) at room temperature for 20 min. Then chromogenic agents from the DAB chromogenic agent kit (Wuhan Boshide Biological Engineering Co., Ltd., Wuhan, China) were added to the sections. Finally, the sections were counterstained with Harris hematoxylin, dehydrated and sealed. Positive LEP-R expression was noted when the membrane presented brown-yellow staining. Sections were observed under a light microscope, and the integral optical density (IOD) of each field was measured with Image-ProPlus analysis software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). The mean IOD value of five fields is reported as the IOD of the section.
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cultured cells or tissues using RIPA lysis buffer (Beyotime, Jiangsu, China), and protein concentrations were measured with the bicinchoninic acid method (BCA, Pierce, Rockford, IL, USA). Equal amounts of protein (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Amersham Bioscience, USA). Membranes were incubated with primary antibody against RUNX2 and GAPDH (both from Santa Cruz Biotechnology, California, USA) at 1:1000 dilution, followed by horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology) at 1:5000 dilution. The protein signal bands were detected using an enhanced chemiluminescence detection reagent (ECL; Thermo Scientific, Rockford, IL, USA).
4
Quantification of RIZ1 Protein Levels in Tumor Tissues
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The RIZ1 protein level was analyzed in 20 paired tumor tissues and non-tumor tissues using western blot. Total protein was extracted with RIPA buffer (Invitrogen). Thirty microgram protein was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride membrane (Millipore Company, Billerica, MA, USA). After blocking with 5% skimmed milk in tris-buffered saline with Tween 20 (TBST), the membrane was incubated with primary antibodies targeting RIZ1 (1:500, cat. AM1194A, Abgent, San Diego, CA, USA) or β-actin (WL0001, Wanlei Bio, Shenyang, China) in TBST containing 1% bovine serum albumin (BSA) overnight at 4°C, followed by incubation with the horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were visualized by an ECL plus chemiluminescence kit (Beyotime Institute of Biotechnology, Haimen, China). β-actin served as a loading control.
5
Western Blot Analysis of JAK-STAT Signaling
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SU-DHL-2/4 cells or tumor tissues were lysed using sodium dodecyl sulfate buffer containing proteinase inhibitors (Roche). Equal amounts of protein (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Shanghai, China). The membranes were blocked and incubated with specific antibodies overnight at 4 °C. The antibodies used were JAK2, p-JAK2, STAT3, p-STAT3, p-Akt, Akt, cyclin D2, P27, IL-6, and GAPDH. The membranes were then incubated with horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology). The protein bands were visualized using an enhanced chemiluminescence reagent.
6
Platelet Activation Signaling Pathway
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Thrombin, 2-methylthio-ADP (2-MeSADP), apyrase (type V), MRS2179 (a P2Y1 receptor antagonist [2 (link)]), prostaglandin E1 (PGE1), sodium citrate, and acetylsalicylic acid were purchased from Sigma (USA). Hexapeptides SFLLRN and AYPGKF were custom synthesized by Invitrogen (USA). Anti-phospho-Akt (Ser473) and anti-β-actin antibodies were purchased from Cell Signaling Technology (USA). Horseradish peroxidase-labeled secondary antibody was obtained from Santa Cruz Biotechnology (USA). Bisindolylmaleimide I (GF 109203X) was purchased from Calbiochem (USA). The AR-C69931MX was a gift from AstraZeneca (UK). All other reagents were reagent grade, and deionized water was used throughout.
7
Collagen-Induced Platelet Activation Assay
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Collagen was purchased from Chrono-log Corporation (Havertown, PA, USA). The 2-MeSADP, thrombin, daphnetin, apyrase (type V), prostaglandin E1 (PGE1), sodium citrate, and acetylsalicylic acid (ASA) were bought from Sigma (St. Louis, MO, USA). Anti-phospho-cPLA2 (Ser505), anti-cPLA2, anti-phospho-ERK (Thr202/Tyr204), anti-ERK, Anti-phospho-AKT (Ser473), and anti-AKT antibodies were from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-labeled secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TxB2 ELISA kit was from Enzo Life Sciences (Exeter, UK). All additional chemicals were of reagent grade.
8
Western Blot Analysis of Transfected Cells
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Transfected PEF cells were harvested by using a cell scraper after washing two times with DPBS. Harvested cells were lysed by sonication in RIPA lysis buffer (Biosesang, Korea) supplemented with protease inhibitor (Roche Applied Science) and centrifuged at 16,000 × g for 5 min at 4℃ to remove cell debris. Protein concentration was determined by using a BCA Protein Assay Kit (Bio-Rad Laboratories, USA). Equal amounts (80 µg) of protein were separated by using a 12% SDS-PAGE gel and then transferred to a polyvinylidene difluoride membrane (No. GF10600023; Millipore, Germany). Subsequently, the membranes were incubated separately with mouse anti-FLAG M2 primary antibody, rabbit anti-β-actin (13E5) monoclonal antibody (Cell Signaling Technology, USA), and rabbit anti-p53 polyclonal antibody or rabbit anti-p21 (Abcam, UK) polyclonal antibody (Abcam) overnight at 4℃. After washing three times with 1× Tris-buffered saline with Tween 20 (LPS Solution), the membranes were incubated with horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology, USA). Chemiluminescent detection was performed by using the ECL reagent (GE Healthcare Life Sciences, USA) and a luminescent image analyzer system (LAS-3000; Fujifilm, Japan).
9
Protein Extraction and Western Blot
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Total protein from approximately 1×106 cells were extracted using radioimmunoprecipitation assay buffer and then quantified using the bicinchoninic acid method (Beyotime, Shanghai, China). Equal amounts of protein sample were separated using 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in TBST (Abcam, Cambridge, UK) for 1 h at room temperature and incubated with primary antibody (Abcam) at 4°C overnight. Membranes were then washed three times andincubated with horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. Signals were detected using an enhanced Chemiluminescent Western Blot Analysis Kit (Thermo Fisher Scientific, Inc.).
10
Profiling Fibroblast Secretome by Western Blot
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Western blot analysis of fibroblast lysates and supernatants was done as previously described [11 (link)]. The following antibodies were used: Anti-COL22A1 (Novus, Littleton, CO, USA), fibronectin, collagen 1A1, CTGF, GAPDH (Santa Cruz, Dallas, TX, USA), ACTA2 (Sigma-Aldrich), and horseradish peroxidase-labeled secondary antibody (Santa Cruz). Signals were detected by chemiluminescence (ProteinSimple, San Jose, CA, USA).
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