Tissue was lysed in radio-immuno-precipitation assay buffer (RIPA) (Boston BioProducts, Ashland, MA), 40ug were fractionated by sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE) using 3%−8% Tris-Acetate gels (NuPage Novex Mini Gel), and the protein was transferred to polyvinylidene diflouride membranes (PVDF) (Millipore, Billerica, MA) and incubated overnight at 4 degrees C with primary antibodies against Transforming growth factor β (TGF-β), Janus family of tyrosine kinase-2 (Jak2), STAT 3, matrix metallopeptidase-9 (MMP-9), SMAD2/3, monocyte chemoattractant protein-1 (MCP-1), α-tubulin, N- Cadherin, α-fodrin, desmin, connexin 43, pan keratin, ß-tubulin, Troponin I, vimentin, filamin, ß-Actin and troponin T (Cell Signaling, Danvers, MA). The following day, membranes were incubated with the appropriate horseradish peroxidase (HRP)-linked secondary antibody for 1h at room temperature (Jackson ImmunoResearch, West Grove, PA). Immune complexes were visualized with enhanced chemi luminescence, images were captured with a digital camera system (G-Box, Syngene, Cambridge, England), and band densitometry was quantified as arbitrary light units using Image J Software (National Institutes of Health, Bethesda, MD). Anti-GAPDH antibody (Cell Signaling) was used on all membranes to correct for loading error. Representative images have been included in the manuscript.
Horseradish peroxidase hrp linked secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase (HRP)-linked secondary antibodies are enzyme-conjugated antibodies used in various immunodetection techniques. HRP is an enzyme that catalyzes the conversion of specific substrates, generating a colored or luminescent signal that can be detected and quantified. These secondary antibodies bind to primary antibodies, allowing for the visualization and analysis of target proteins or other biomolecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Boston BioProducts (1 mentions)
G box, by Syngene (1 mentions)
Desmin, by Cell Signaling Technology (1 mentions)
Pan keratin, by Cell Signaling Technology (1 mentions)
Connexin 43, by Cell Signaling Technology (1 mentions)
α fodrin, by Cell Signaling Technology (1 mentions)
Troponin 1, by Cell Signaling Technology (1 mentions)
Mcp 1, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate kit, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Merck Group (1 mentions)
Gel doc system, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
7 protocols using horseradish peroxidase hrp linked secondary antibody
1
Protein Expression Analysis in Tissue Lysates
2
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and lysed using RIPA lysis buffer (Beyotime, Haimen, China) containing 1 mM PMSF and 1× protease inhibitor cocktail (Sigma-Aldrich, St. Louise, MO, USA). Cell lysates were separated using SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were blocked in Tris-buffered saline containing 0.01% Tween20 and 5% non-fat milk for 1 h and then incubated with specific primary antibodies. Following incubation with horseradish peroxidase (HRP)-linked secondary antibody (Jackson, West Grove, PA, USA) at room temperature for 1 h, detection was performed using an Immobilon Western Chemiluminescent HRP substrate kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The primary antibodies used were as previously described.
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed western blot assay as shown previously [70 (link)]. In brief, cells or tissues were lysed on ice with RIPA lysis buffer, including 1% protease (Roche) and 1% phosphatase inhibitor cocktail (Sigma). We quantified protein samples using BCA assay (Sigma), and separated proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred proteins onto polyvinylidene fuoride (PVDF) membranes (Millipore). The membranes were blocked with 5% milk, and incubated with indicated primary antibodies, then probed with horseradish peroxidase (HRP)-linked secondary antibodies (Jackson ImmunoResearch, PA, USA). We used ECL for signal detection and western blot images were acquired with Bio-Rad GelDoc system.
4
Investigating Fatty Acid Metabolism Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Modified Eagle’s medium (MEM), trypsin, fetal bovine serum (FBS), glutamine, and antibiotics were purchased from Dutscher Laboratories. All purified FA (palmitic acid, (PAL), DHA, EPA, α-linolenic acid (LNA), oleic acid (OA), arachidonic acid (AA), and linoleic acid (LA)) and FA-free bovine serum albumin (BSA), thapsigargin, and 4-phenyl butyrate acid (PBA) were purchased from Sigma Aldrich (Saint Quentin Fallavier, France). The antibodies raised against CCAAT-enhancer-binding protein homologous protein (CHOP) and activating transcription factor 4 (ATF4) were purchased from Cell Signaling Technology (Ozyme, France). The horseradish peroxidase (HRP)-linked secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (Interchim, France). The antibody raised against β-actin (C4) HRP was purchased from Santa Cruz. The Muc2 Elisa Kit (Human) was purchased from Aviva Systems Biology (Clinisciences, Nanterre, France).
5
Quantitative Immunoblot Analysis of Thioredoxin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum samples were diluted with 1×PBS into 5 mg/ml. The cell lysates were adjusted to a protein concentration of 2 mg/ml. Serum samples (200 μg/lane) or the cell lysates (80 μg/lane) were resolved by SDS-PAGE (10% gel), then transferred onto a PVDF membrane, followed by blocking with 5% (w/v) nonfat dry milk in 50 mM Tris, pH 7.6, 150 mM NaCl and 0.1% Tween-20 (TBS-T) for 2 ~ 4 h at room temperature. Antibodies were diluted with TBS-T containing 5% nonfat dry milk. The membrane was probed with corresponding primary antibody overnight at 4°C. Antibodies of Trx1 and TrxR1 were used at 1:500 dilution, respectively. Since Trx1/TrxR1 can be secreted by cells, this study thus payed more attention to Trx1/TrxR1. Horseradish peroxidase (HRP)-linked secondary antibodies (Jackson, USA) was used at a 1:10000 dilution and incubated with membranes for 2 h at room temperature. The protein bands were visualized using enhanced chemiluminescence (ECL) system, followed by exposure to Amersham Hyperfilm MP at room temperature. The immunoblot bands were analyzed using semi-quantitatively using ImageJ 1.42q.
6
Western Blot Analysis of Ribosomal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All samples were resolved on 12 or 14% (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 4% Milk in Tris buffered saline, pH 7.4 containing 0.1% Tween-20 (TBS-T) for 1 hour at room temperature. Primary antibodies [Actin (1:1,000, #SC-1616-G, Santa Cruz), HA (1:1,000, #3724, Cell Signaling), Fasn (1:1000, #3189, Cell Signaling), Acot1 (1:1000, #ab100915, Abcam), Rps2 (1:1000, #A303-794, Bethyl Labs), Rpl36 (1:1000, #ab241584, Abcam), Rps17 (1:1000, #ab128671, Abcam)] were diluted in 3% bovine serum albumin (BSA) and incubated overnight at 4°C. The following day, membranes were washed and incubated with the appropriate horseradish peroxidase (HRP)-linked secondary antibodies (1:10,000, Jackson ImmunoResearch) in 3% BSA followed by visualization with a chemiluminescent substrate (Thermo Scientific). Western blot quantification was performed by densitometric analysis using ImageJ (NIH). The signal intensity in arbitrary units was determined for HA protein in the IP and the amount of Actin in the supernatant. After normalization to Actin, HA protein expression in RiboHET;ChATCre and RiboHO;ChATCre mice was displayed relative to wild-type controls.
7
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CNE-2 cells were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) and supplemented with protease inhibitor cocktail kit (Roche Biochemicals, Penzberg, Germany). Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The membrane was incubated with primary antibodies for LMP1- (1:2000, Abcam, Cambridge, UK), LMP2A- (1: 1000, Abcam), eIF4E-specific (1: 1000, Abcam) or β-actin (1: 3000, Sino Biological, Wayne, PA, USA). Horseradish peroxidase (HRP)-linked secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and enhanced chemiluminescence (Thermo Fisher Scientific) were utilized to visualize the specific binding of the HRP-linked second antibody to first antibody-protein complexe, under the UVP BioSpectrum 500 imaging system (UVP, Upland, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!