Ab13867

Manufactured by Abcam

Sourced in United States, United Kingdom, China

Ab13867 is a primary antibody product offered by Abcam. It is a recombinant monoclonal antibody targeting a specific protein. The antibody is validated for use in various immunoassay techniques.

Automatically generated - may contain errors

Lab products found in correlation

Bca kit, by Thermo Fisher Scientific (9 mentions) Ab16502, by Abcam (5 mentions) Ab86299, by Abcam (5 mentions) Ab6721, by Abcam (3 mentions) Ab32536, by Abcam (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Ripa buffer, by Beyotime (2 mentions) Ab181602, by Abcam (2 mentions) Ab33915, by Abcam (2 mentions) Ab9485, by Abcam (2 mentions) Ab18256, by Abcam (2 mentions) Ab32503, by Abcam (2 mentions) Ripa lysis buffer, by Beyotime (2 mentions) Bca protein assay, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Ab24182, by Abcam (1 mentions) Protein a g beads, by Abcam (1 mentions) Ab16502, by Santa Cruz Biotechnology (1 mentions) Ab23875, by Abcam (1 mentions) Ab3759, by Abcam (1 mentions)

31 protocols using ab13867

Immunohistochemistry and Immunofluorescence Analysis of TLR4, p65, and pp65

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Immunohistochemistry was performed as previously reported.²³, ³⁹, ⁴⁰ In brief, the skin tissue fixed in 10% formalin buffer was embedded in paraffin blocks and cut into 4 μm‐thick tissue sections. The processed tissue sections were then dewaxed and treated with 3% H₂O₂ for 15 minutes, followed by blocked with goat serum for 30 minutes, incubated at 4°C overnight with a primary antibody against TLR4 (ab13867, 1:300, Abcam), p65 (ab32536, 1:200, Abcam) and immunostained with a SP‐9000 HistostainTM Kit (SP‐9000D, ZSGB), according to the manufacturer's instructions.
For immunofluorescence analysis,²³, ³⁹ cells were fixed in 4% formaldehyde for 30 minutes, washed with phosphate‐buffered saline (PBS), permeabilized with 0.1% Triton‐X100 for 10 minutes at room temperature, blocked with 1% bovine serum albumin (BSA), hybridized with a antibody specific for TLR4 (ab13867, 1:300, Abcam), p65 (ab32536, 1:200, Abcam) and pp65 (ab86299, 1:200, Abcam) at room temperature for 1 hours and then incubated with a Cy3‐conjugated goat secondary antibody (cw0159, 1:100, Cwbio, China) at 37°C for 1 hours. Finally, the samples were stained with 4′,6′‐diamidino‐2‐phenylindole (DAPI, Sigma).

Shi J., Shi S., Xie W., Zhao M., Li Y., Zhang J., Li N., Bai X., Cai W., Hu X., Hu D., Han J, & Guan H. (2021). IL‐10 alleviates lipopolysaccharide‐induced skin scarring via IL‐10R/STAT3 axis regulating TLR4/NF‐κB pathway in dermal fibroblasts. Journal of Cellular and Molecular Medicine, 25(3), 1554-1567.

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Immunoprecipitation of Protein Complexes

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HEK293T cell expressing the proteins of interest in 6-well plates were washed with 1x PBS and lysed with 200 μL HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% NP-40 pH 7.5), supplemented 100X protease inhibitor cocktail (Calbiochem) at 4°C for 1 h and subsequent lysate clearance by centrifugation at 16,000 g at 4°C for 10 min. In case of immunoprecipitation of purified proteins, 2μg of each target protein, (5 μg of GP proteins) mixed with HEPES lysis buffer to a final volume of 20 μL. For cell-free immunoprecipitation purified GP, and commercially available pure MD2 (ab238343, Abcam) and TLR4 (ab233665, Abcam) were incubated with 2 μL anti-MD2 (ab24182, Abcam), anti-TLR4 (ab13867, Abcam) and anti-His (51-9000012, BD-Pharmagen) antibodies at 4°C for 4 h followed by addition of 20 μL protein A/G beads (Abcam) and further 2 h incubation. The beads were washed with HEPES lysis buffer and eluted with 1 M Glycine pH 3 followed by NaOH neutralization.

Scherm M.J., Gangloff M, & Gay N.J. (2022). Activation of Toll-like receptor 4 by Ebola virus-shed glycoprotein is direct and requires the internal fusion loop but not glycosylation. Cell Reports, 41(4), 111562.

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Western Blot Analysis of Signaling Proteins

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Protein concentration from cells and islet tissue was determined by bicinchoninic acid protein assays. Protein samples were separated by SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Sigma). Membranes were blocked with 5% fat-free milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 and incubated overnight at 4°C with anti-TLR4 (1 : 1000, Abcam, UK, ab13867), anti-NF-κB p65 (1 : 1000, Abcam, UK, ab16502), anti-GRP40 (1 : 200, Santa Cruz, sc-32905), anti-PLC (1 : 500, Abcam, UK, ab243181), anti-pAMPKα1 (1 : 1000, Abcam, UK, ab23875), anti-AMPK (1 : 500, Abcam, UK, ab3759), or anti-β-actin antibody (1 : 200, Abcam, UK, ab8226). Membranes were then incubated with horseradish peroxidase-labeled secondary antibody (1 : 500) for 1 hour at room temperature. Protein signals were visualized using the enhanced chemiluminescence detection system.

Shen X., Fan B., Hu X., Luo L., Yan Y, & Yang L. (2019). Metformin Reduces Lipotoxicity-Induced Meta-Inflammation in β-Cells through the Activation of GPR40-PLC-IP3 Pathway. Journal of Diabetes Research, 2019, 7602427.

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Quantitative Protein Expression Analysis

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Harvested cells or collected tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Shanghai, China) with protease inhibitor (Sigma-Aldrich, Darmstadt, Germany). The protein was quantified using the bicinchoninic acid (BCA) protein assay (Thermo Scientific, Waltham, MA, USA). Total proteins were resolved by 12% Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis and were transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). Membranes were blocked with 5% nonfat dry milk in PBST for 1 h and then incubated with primary antibodies against mouse Fibronectin (ab45688, Abcam), TLR4 (ab13867, Abcam), NF-κB (#8242, CST), phospho-NF-κB p65 (#3031, CST), MyD88 (#4283, CST) or GAPDH (#5174, CST) followed by incubation with a peroxidase-conjugated goat anti-rabbit (or mouse) IgG antibody. Densitometry analysis was performed, and the results were normalized to GAPDH expression and expressed as the fold changes over controls.

Chen L., Sha M.L., Li D., Zhu Y.P., Wang X.J., Jiang C.Y., Xia S.J, & Shao Y. (2017). Relaxin abrogates renal interstitial fibrosis by regulating macrophage polarization via inhibition of Toll-like receptor 4 signaling. Oncotarget, 8(13), 21044-21053.

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Western Blot Analysis of Immune Signaling

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Total proteins were isolated with RIPA buffer, separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. The membrane was blocked and incubated with primary antibodies and secondary antibodies in order. Immobilon western chemilum HRP substrate (Millipore, MA, USA) was used to visualize the protein bands. Primary antibodies used were anti-GAPDH (ab181602, Abcam), anti-TLR4 (ab13867, Abcam), anti-NF-κB p65 (ab16502, Abcam), anti-NF-κB p65 (phospho S536) (ab86299, Abcam), anti-IRAK4 (ab5985, Abcam), anti-IRAK4 (phospho T345) (ab216513, Abcam), anti-IL-6R (ab128008, Abcam), anti-STAT3 (ab119352, Abcam), anti-STAT3 (phospho Y705) (ab128008, Abcam), anti-cleaved caspase-3 (ab2302, Abcam), anti-Bcl-2 (ab182858, Abcam), and anti-cleaved PARP1 (ab32561, Abcam).

Shi C., Zhao Y., Li Q, & Li J. (2021). lncRNA SNHG14 Plays a Role in Sepsis-Induced Acute Kidney Injury by Regulating miR-93. Mediators of Inflammation, 2021, 5318369.

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Hippocampal TLR4 and TNF-α Quantification

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Mice were decapitated 24 hr after the behavioral tests, and brains were rapidly removed. According to the mouse brain map (Paxinos & Franklin, 2004), the hippocampus was dissected on ice and placed into liquid nitrogen to be frozen and then stored at −80°C until use. Proteins from hippocampus were dissolved in RIPA lysis buffer (CW Biotech), and their concentration was measured using BCA Protein Assay Kit (CW Biotech). Total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) in reducing conditions using 12% or 8% acrylamide gels and analyzed by immunoblotting using anti‐TLR4 (1:1,000; ab13867; Abcam), anti‐TNF‐α (1:1,000; ab66579; Abcam), and anti‐β‐actin (1:10,000, #3700; CST) as primary antibodies. Band intensity was semi‐quantitatively analyzed using the Quantity One software (Bio‐Rad), and the expression level of TLR4 and TNF‐α was normalized to that of β‐actin.

Zhang K., Lin W., Zhang J., Zhao Y., Wang X, & Zhao M. (2020). Effect of Toll‐like receptor 4 on depressive‐like behaviors induced by chronic social defeat stress. Brain and Behavior, 10(3), e01525.

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Western Blot Analysis of TLR-4 Modulation

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Western blot analysis TLR-4 expression and its modulation on LPS administration were performed by standard methods. In brief, the whole tissue was lysed with RIPA-NP40 and 60 μg of the tissue lysate was separated on an 10% sodium dodecyl sulphate- (SDS-) polyacrylamide gel and blotted onto nitrocellulose membrane. The membranes were blocked with 5% BSA in TBST for 3 hour at room temperature, washed and incubated with primary anti-mice TLR-4 Abs in 1/1000 dilution (cat no- ab13867, Abcam, UK, synthetic peptide corresponding to amino acid 39–56 of mouse TLR-4) overnight at 4°C. The membranes were washed with TBST and incubated with the appropriate HRPO-conjugated secondary antibody in 1/5000 dilution (cat no-ab6721, Abcam, UK) for 1 h at room temperature. Detection of antigen was performed using the enhanced chemiluminescent detection method (ECL-plus cat no-ab140357 Abcam, UK). We have used Beta-tubulin as loading control for western blot to ensure equal loading throughout the gel as it is a housekeeping gene that exhibit high-level, constitutive expression in the sample. It also has a different molecular weight than our protein of interest that is, TLR-4, to help distinguish between both bands [14 (link), 46 (link)].

Ghosh C, & Bishayi B. (2015). Characterization of Toll-Like Receptor-4 (TLR-4) in the Spleen and Thymus of Swiss Albino Mice and Its Modulation in Experimental Endotoxemia. Journal of Immunology Research, 2015, 137981.

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Western Blot Analysis of TLR4 Expression

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Proteins were extracted from hPASMC cells using RIPA buffer (Beyotime Biotechnology Shanghai, China), separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Inc., Bedford, MA, USA). The membranes were incubated with solution containing 5% skim milk powder for 2 h at room temperature and then with anti-TLR4 (1: 1000; ab13867, Abcam, Cambridge, MA, USA) and anti-β-actin (1: 1000; ab179467, Abcam) at 4°C for 12–16 h. The membranes, were washed and incubated with secondary antibody for 2 h at 26°C, followed by incubation with an enhanced chemiluminescence (ECL) reagent. The expression of TLR4 relative to β-actin was analyzed in triplicate samples.

He M., Shen J., Zhang C., Chen Y., Wang W, & Tao K. (2020). Long-Chain Non-Coding RNA Metastasis-Related Lung Adenocarcinoma Transcript 1 (MALAT1) Promotes the Proliferation and Migration of Human Pulmonary Artery Smooth Muscle Cells (hPASMCs) by Regulating the MicroRNA-503 (miR-503)/Toll-Like Receptor 4 (TLR4) Signal Axis. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e923123-1-e923123-14.

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Immunohistochemical Analysis of Lung Inflammation

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The protein expression levels of TLR4, IL-1R1, ST2, and SIGIRR were assessed in rat lung tissues by IHC using a kit from DakoCytomation (Carpinteria, CA). In brief, the tissue samples were fixed with paraformaldehyde and embedded in paraffin. After washing in PBS, the slides were blocked with a protein-block solution for 20 min and incubated overnight with primary antibodies against TLR4 (Abcam, ab13867), IL-1R1 (Abcam, ab40774), ST2 (SCBT, sc18687), and SIGIRR (SCBT, sc98976) at dilutions of 1:200, 1:200, 1:100, and 1:100 in blocking solution, respectively. The slides were washed and incubated with biotinylated link universal antiserum, followed by horseradish peroxidase-streptavidin conjugate. The slides were rinsed and the color was developed using 3,3-diaminobenzidine hydrochloride as a chromogen. Finally, the sections were rinsed in distilled water, counterstained with Mayer’s hematoxylin solution and mounted with DPX mounting medium for evaluation.
For histopathological examination, the paraffin-embedded tissues were stained with hematoxylin & eosin (H & E) stain. The slides were evaluated for morphologic changes and pathological signatures of ischemia, inflammation, and any other abnormalities. The sections were observed with Olympus microscope IX701, and digital computer images were recorded using an Olympus DP70 camera.

Yadav V.R., Vilekar P., Awasthi S, & Awasthi V. (2014). Hemorrhage-inducedIL-1R pathway in lung is suppressed by 3,5-bis(2-fluorobenzylidine)-4-piperidone (EF24) in a rat model of hypovolemic shock. Artificial organs, 38(8), 675-683.

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Metabolic Profiling and Histological Analysis

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Plasma insulin and hepatic triglyceride and cholesterol were measured using commercial kits. Glucose tolerance test (GTT), insulin tolerance test (ITT) and histological analyses, including hematoxylin & eosin (H&E) and Alcian blue-periodic acid Schiff (AB-PAS) staining, immunohischemistry of TLR4 (ab13867, Abcam), F4/80 (GB11027, Servicebio), and UCP1 (ab155117, Abcam), immunofluorescence staining of LC3 (14600-1-AP, Proteintech), were performed as described previously.^{52 (link)} Adipocyte size was determined from H&E stained sections and the number of CLSs was manually counted from F4/80-stained sections in five random fields from 5 mice per group by Image J software. Intestinal permeability was assessed in vivo following oral administration of fluorescein-isothiocyanate (FITC)-dextran (46944–500 MG-F, Sigma). Fecal SCFAs levels were detected by GC/MS (Thermo, TRACE 1310-ISQ LT), and the final data were normalized according to the fecal weight.

Ma L., Ni Y., Wang Z., Tu W., Ni L., Zhuge F., Zheng A., Hu L., Zhao Y., Zheng L, & Fu Z. (2020). Spermidine improves gut barrier integrity and gut microbiota function in diet-induced obese mice. Gut Microbes, 12(1), 1832857.

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