Ab2068

Manufactured by Abcam

Sourced in United States

Ab2068 is a primary antibody designed for use in immunoassays. It is a rabbit polyclonal antibody that recognizes a specific target antigen. The core function of this product is to bind to and detect the target antigen in samples.

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Lab products found in correlation

4 protocols using ab2068

Hippocampal TLR4-Myd88-NF-κB Signaling

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The hippocampal tissue was grid and centrifuged in a pre-chilled tissue lysate at 12,000 rpm for 30 min. The supernatant of total protein was extracted for SDS-PAGE and the protein was semi-dried for membrane transfer. The cells were blocked 2 h and incubated overnight at 4°C with TLR4 (ab22048, Abcam, Cambridge, MA, USA), Myd88 (ab2068, Abcam, USA), NF-κB antibody (ab32360, Abcam, USA), followed three washes and incubation with secondary antibody for 1 h. After four washes using TBST, cells were developed with ECL Western Blotting Substrate kit (32109, Pierce™, Thermo Fisher Scientific, Waltham, MA, USA) and gray value was measured by using Quantity One software.

Chen K., Sun Y., Diao Y., Ji L., Song D, & Zhang T. (2017). α7 nicotinic acetylcholine receptor agonist inhibits the damage of rat hippocampal neurons by TLR4/Myd88/NF-κB signaling pathway during cardiopulmonary bypass. Molecular Medicine Reports, 16(4), 4770-4776.

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Immunohistochemical Analysis of TLR4 Signaling

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Formalin-fixed paraffin sections were deparaffinized in xylene and rehydrated through an ethanol series. The sections were then immersed in 0.3% hydrogen peroxidase for 15 min to deplete endogenous peroxidase and exposed to high pressure to activate their antigenicity. Sections were then incubated with blocking solution (PBS, 3% of BSA, 0.1% Triton-X 100) at room temperature for 30 minutes. The primary antibody of TLR4 (1:20, ab22048; Abcam), MD2 (ab24182; Abcam), Myd88 (ab2068; Abcam), NF-κB (ab7970; Abcam), IL-6 (ab6672; Abcam), TGF-β (ab66043; Abcam), VEGF (ab1316; Abcam), EGF (ab115562; Abcam), and MMP2 (ab86607; Abcam) were added and incubated at 4 °C overnight. Sections were washed with PBS, incubated with FITC–conjugated goat anti-mouse secondary antibody at room temperature for 1 hour (1:400), and washed three times with PBS at room temperature for 10 minutes. Images were obtained with a fluorescence microscope (OLYMPUS DP70; Olympus Co., Tokyo, Japan).

Guo J., Chen L., Luo N., Li C., Chen R., Qu X., Liu M., Kang L, & Cheng Z. (2016). LPS/TLR4-mediated stromal cells acquire an invasive phenotype and are implicated in the pathogenesis of adenomyosis. Scientific Reports, 6, 21416.

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In Vitro Stimulation of Inflammatory Response

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ISOF and FSK (purity 99%) were purified as white crystal by Kunming Baker Norton Pharmaceutical Co., Ltd. DM injection was purchased from Yangtze River Pharmaceutical Company (Yangzhuo, Zhejian, China). LPS (

Escherichia coli

055:B5),
dimethyl sulfoxide (DMSO; 0227), trypan blue powder (72571), and roflumilast (SML1099) were purchased from Sigma‐Aldrich Co. (St Louis, MO). Sterile water for injection (H41024923) was purchased from Sinopharm Group Health and Pharmaceutical Co. (Tianjin, China). Protein chip assay kit (QAH‐TH17‐1) was purchased from RayBiotech, Inc. (Guangzhou, China). Enzyme‐linked immunosorbent assay (ELISA) kit for IL‐1β (VAL 101, the minimum detectable dose of IL‐1β is typically less than 1.0 pg/ml) and TNF‐α (VAL105, the minimum detectable dose of TNF‐α is typically less than 7.8 pg/ml) were purchased from R&D Systems, Inc. (Minneapolis, MN). Mouse TLR4 polyclonal antibody (ab13556), rabbit MYD88 polyclonal antibody (ab2068), rabbit NF‐κB (P65) antibody (ab7970), and anti‐beta actin antibody (ab49900) were purchased from Abcam Company (USA). Rabbit GAPDH monoclonal antibody (2118) was purchased from Cell‐signaling Technology Inc (Massachusetts, USA). Lymphocyte separation solution (LTS1007N) and Human Blood preservation solution were purchased from Tianjin Haoyang Biological Products Technology Co., Ltd (Tianjin, China).

Du X., Shi R., Wang Y., Wu W., Sun S., Dai Z., Chen C., Weng Z., Li X., Liu Q., Zhang L., Saidian M, & Yang W. (2019). Isoforskolin and forskolin attenuate lipopolysaccharide‐induced inflammation through TLR4/MyD88/NF‐κB cascades in human mononuclear leukocytes. Phytotherapy Research, 33(3), 602-609.

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Protein Extraction and Western Blot Analysis

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The protein extraction kit (P0013B, Beyotime, Shanghai, China) was applied to extract the proteins of RECs and then a BCA protein determination kit (P0012, Beyotime, Shanghai, China) was used to measure protein concentration. Proteins’ fractions were then separated on SDS-PAGE electrophoresis gels and transferred to PVDF membranes (IPVH00010, Millipore, Boston, MA, USA), which were blocked by using 5% skim milk powder for 2 h. Membranes were blocked, then incubated at 4 °C overnight with multiple primary antibodies, including TLR4, MyD88, P-NF-κB p65 (ab22048, ab2068, ab86299, Abcam, Cambridge, UK), NF-κB p65 (AF5006, Affinity Biosciences, Jiangsu, China), P-IκBα, IκBα, P-JNK, JNK, P-ERK, ERK, P-p38 MAPK, p38 MAPK (9246, 4814, 9251, 9252, 4370, 4695, 4511, 8690, Cell Signaling Technology, Danvers, MA, USA), and β-actin (c-47778, Santa Cruz, Dallas, TX, USA), respectively. After that, secondary antibodies were incubated on the membranes for 45 min, which were detected by enhanced chemiluminescence (ECL) system (5600, Tanon, Shanghai, China) and analyzed using the ImageJ software (Media Cybernetics, Bethesda, MD, USA).

Zhao C., Yi F., Wei B., Tan P., Huang Y., Zeng F., Wang Y., Xu C, & Wang J. (2023). Sodium Propionate Relieves LPS-Induced Inflammation by Suppressing the NF-ĸB and MAPK Signaling Pathways in Rumen Epithelial Cells of Holstein Cows. Toxins, 15(7), 438.

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