To determine the TLR4, MyD88 phosphorylated (p)-NF-κB, TRIF3 and IRF3 protein levels in the lung tissue, western blot analysis was performed as described by Zhang et al (12 (link)). Primary antibodies (all from Abcam, Cambridge, UK) were used at the indicated dilutions as follows: Rabbit polyclonal to TLR4 (cat. no. ab13556; 1:500); rabbit polyclonal to MyD88 (cat. no. ab2064; 1:1,000); rabbit polyclonal to NF-κB p65 (cat. no. ab7970; 1:1,000); rabbit polyclonal to p65-NF-κB (cat. no. ab7970; 1:500); rabbit polyclonal to TRIF (cat. no. ab13810; 1:500); rabbit monoclonal to IRF3 (cat. no. ab68481; 1:1000). Proteins (30 µg) were run on a 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. Following incubation with 10% non-fat milk for 1 h, the membranes were probed with primary antibodies overnight at 4°C and then incubated with HRP-labeled goat anti-rabbit secondary antibodies (1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The protein levels were normalized using β-actin as a loading control. The relative optical density of the protein bands was measured using a Zeineh Laser Densitometer (Biomed Instruments, Inc., Fullerton, CA, USA) after subtracting the film background.
Hrp labeled goat anti rabbit secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
HRP-labeled goat anti-rabbit secondary antibodies are laboratory reagents designed to detect and visualize the presence of rabbit primary antibodies in various immunoassay techniques. These secondary antibodies are conjugated with horseradish peroxidase (HRP), an enzyme that can catalyze a colorimetric or chemiluminescent reaction, allowing for the identification and quantification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one analysis software, by Bio-Rad (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Beyotime (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Mouse anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Clarity max western ecl substrate, by Bio-Rad (1 mentions)
Mouse anti egfp, by Santa Cruz Biotechnology (1 mentions)
4 protocols using hrp labeled goat anti rabbit secondary antibody
1
Lung Tissue Protein Quantification
2
Quantitative Analysis of β-Catenin Protein
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Following transfection, the cells were harvested and immediately prepared for protein extraction. The protein content in the supernatant was detected using the bicinchoninic acid method (Pierce, Rockford, IL, USA). Equal quantities of protein were run on 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. Following incubation with 10% non-fat milk for 1 h, the membranes were probed with polyclonal rabbit anti-rat β-catenin antibody (1:400; Sigma, St. Louis, MO, USA) overnight at 4°C and then incubated with HRP-labeled goat anti-rabbit secondary antibodies (diluted 1:3,000; Santa Cruz Biotechnology, Inc.). The protein levels were normalized using β-actin as a loading control. The relative optical density of the protein bands was measured using a Zeineh Laser Densitometer (Biomed Instruments Inc., Fullerton, CA, USA) after subtracting the film background.
3
Western Blot Analysis of Lung Proteins
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In lung homogenates, the expression of PRMT6, H3R2me2a, and H3K4me3 in mouse and human samples, as well as Bcl-2, Bax, and endothelial nitric oxide synthase (eNOS) in mouse samples were detected by standard Western blotting techniques. Homogenate supernatants were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology, Shanghai, China), transferred onto polyvinylidene fluoride membranes, blocked for 1 hour, then washed, incubated with rabbit monoclonal antibodies against PRMT6, H3R2me2a, H3K4me3, Bcl-2, Bax, and eNOS (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The next day, the membrane was washed and incubated for 2 hours at 37°C with HRP-labeled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology Inc, Dallas, TX, USA), and then washed again. Labeled proteins were visualized by the electrochemiluminescence plus Western blotting detection system (Immobilon-P; EMD Millipore, Billerica, MA, USA). Band densities were quantified using Quantity One Analysis Software (Bio-Rad Laboratories Inc, Hercules, CA, USA).
4
Quantifying HSV1-TK Protein Expression
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Selected B-cell lines grown to mid-log phase were seeded in 24-well plates at a density of 2 × 105 cells per well. Twenty-four hours later, cells were transduced with an rAAV6.2 serotype encoding HSV1-TK at an MOI of 105. Forty-eight hours after transduction, cells were harvested, lysed in RIPA buffer and quantified by Bradford assay. Total cellular protein (20 µg per lane) was separated by NuPAGE Bis–Tris (4–12%) polyacrylamide gel electrophoresis (Thermo Fisher Scientific) in MOPS buffer under reducing conditions. Protein was transferred onto a nitrocellulose membrane that was blocked with 5% skimmed milk in TBST for one hour prior to antibody incubation, five membrane washes and final detection with the ECL chemiluminescent detection kit, Clarity Max Western ECL Substrate (Bio-Rad).
The primary antibody rabbit polyclonal anti-HSV1-TK was generously provided by Dr. William Summers of Yale University. HRP-labeled goat anti-rabbit secondary antibody was used for final detection (Santa Cruz Biotechnology).
The primary antibodies, mouse anti-EGFP and mouse anti-GAPDH, were obtained from Santa Cruz Biotechnology, as were HRP-labeled goat anti-mouse IgG secondary antibodies used for chemiluminescent detection.
The primary antibody rabbit polyclonal anti-HSV1-TK was generously provided by Dr. William Summers of Yale University. HRP-labeled goat anti-rabbit secondary antibody was used for final detection (Santa Cruz Biotechnology).
The primary antibodies, mouse anti-EGFP and mouse anti-GAPDH, were obtained from Santa Cruz Biotechnology, as were HRP-labeled goat anti-mouse IgG secondary antibodies used for chemiluminescent detection.
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