Ab219800

Manufactured by Abcam

Sourced in United States, United Kingdom, China

Ab219800 is a lab equipment product. It is a Fluorescein Isothiocyanate (FITC) Conjugated Immunoglobulin G (IgG) Antibody. The core function of this product is to serve as a detection reagent in immunoassays and other applications where a fluorescent antibody is required.

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Anti-GSDMD (ab219800) (2 citations)

73 protocols using ab219800

Histological Assessment of Osteoarthritis

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The knee joints were fixed in 10% neutral buffered formalin at room temperature for 24 h then decalcified in Immunocal (StatLab, McKinney, TX) for 3 days; fresh Immunocal was changed every 24 h. Tissues were processed and paraffin-embedded, then 5-μm-thick sagittal sections were generated, starting from the medial side of the knees. They were stained with Safranin-O. OARSI scoring was based on an established scoring system [39 (link)]. Synovitis scoring was based on the severity of synovium hyperplasia (score range 0–3) and inflammatory cell infiltration (score range 0–3) in the sub-synovial region [40 (link)]. All histological scoring was performed by two blinded reviewers.
For immunohistochemistry, the sections were deparaffinized and rehydrated using xylene followed by an ethanol gradient. Antigen retrieval was performed using citrate buffer (Dako, S1699) at 3 psi. The sections were incubated overnight at 4 °C with primary antibodies against GSDMD (1:200, Abcam, ab219800), followed by 1 h incubation with biotinylated goat secondary antibody (Vector Laboratories, BA-9200). DAB reagent (Vector Laboratories, SK-4100) was used for the peroxidase-substrate reaction.

Yang T., Sun K., Wang C., Swarnkar G., Quan S., Kress D., Xiao J., Alippe Y., Zheng H., Brophy R.H., Hao D., McAlinden A., Abu-Amer Y., Shen J, & Mbalaviele G. (2021). Gasdermin D deficiency attenuates arthritis induced by traumatic injury but not autoantibody-assembled immune complexes. Arthritis Research & Therapy, 23, 286.

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Protein Extraction and Western Blot Analysis of Myocardial Tissue

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The total protein content in the myocardial tissue was extracted using the total protein extraction kits (Nanjing JianCheng Bioengineering Institute, Nanjing, Jiangsu, China), and its concentration was determined by the BCA method (NanJing JianCheng). The samples were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked using 5% skim milk at room temperature for 2 h and then subjected to overnight incubation at 4°C with the primary antibodies (all from Abcam) against TLR4 (dilution ratio of 1:300, ab217274), NLRP3 (dilution ratio of 1:1000, ab214185), caspase‐1 (dilution ratio of 1:1000, ab1872), gasdermin D (GSDMD) (dilution ratio of 1:1000, ab219800), NF‐κB (dilution ratio of 1:1000, ab16502), NF‐κB p65 (phospho S536) (dilution ratio of 1:2000, ab86299) or GAPDH (dilution ratio of 1:1000; ab181602). Next, the membranes were incubated with Tris‐buffered saline‐tween containing 0.1% tween 20 and the secondary antibody IgG H&L‐conjugated horseradish peroxidase (dilution ratio of 1:5000, ab205718). Protein band densities were evaluated using the image‐pro plus version 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

Xuan L., Fu D., Zhen D., Bai D., Yu L, & Gong G. (2022). Long non‐coding RNA Sox2OT promotes coronary microembolization‐induced myocardial injury by mediating pyroptosis. ESC Heart Failure, 9(3), 1689-1702.

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Western Blot Analysis of GSDMD

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Peritoneal macrophages incubated with or without SEZ were treated with RIPA lysis buffer to extract total proteins. Protein concentration was measured using BCA Protein Assay Kit (Beyotime Biotechnology, China). An equal amount of samples were separated by SDS-PAGE. All proteins were subsequently electrotransferred onto the polyvinylidene difluoride (PVDF) membranes and then blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. After washing three times, the PVDF membranes were incubated overnight at 4 °C with the following primary antibodies diluted in BSA: Anti-GSDMD/GSDMD-N antibody (1:1000, ab219800) and Anti-β-Actin antibody (1:1000, ab8227) from Abcam. After washing away remain primary antibodies, the membranes were incubated with second antibodies (Goat Anti-Rabbit IgG H&L, 1:10000, ab205718, Abcam, UK) at room temperature for 1 h, followed by visualization using an ECL reagent (Solarbio, China). Finally, Immunoblot detection was performed by a Gel Imaging System (4200; Tanon, China). All proteins were scanned and quantified by the ImageJ software.

Xu G., Guo Z., Liu Y., Yang Y., Lin Y., Li C., Huang Y, & Fu Q. (2022). Gasdermin D protects against Streptococcus equi subsp. zooepidemicus infection through macrophage pyroptosis. Frontiers in Immunology, 13, 1005925.

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Western Blot Analysis of Inflammasome Proteins

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Western blot was performed as described previously. 24 (link) Specific proteins were detected using the following primary antibodies: NLRP3 (no. ab214185, 1:1000, Abcam), ASC (no. A1170, 1:1000, ABclonal), caspase-1 (no. ab238972, 1:1000, Abcam), IL-1β (no. ab9722, 1:1000, Abcam) and GSDMD (no. ab219800, 1:1000, Abcam). Horseradish peroxidase-conjugated secondary antibodies (ZB-2301, ZB-2305, 1:1000, ZSGB) were incubated with the membrane and the antibody complexes were detected by Imaging System (Bio-Rad, Hercules, CA, USA). β-Actin (no.TA-09, 1:1000, ZSGB) was used as the control to detect equal protein loading.

Yang H., Liu S., Du H., Hong Z., Lv Y., Nie C., Yang W, & Gao Y. (2021). Hydrogen Attenuates Myocardial Injury in Rats by Regulating Oxidative Stress and NLRP3 Inflammasome Mediated Pyroptosis. International Journal of Medical Sciences, 18(14), 3318-3325.

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Inflammasome Pathway Protein Analysis

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Lung tissue and BEAS-2B cells were lysed with RIPA buffers containing PMSF or phosphatase inhibitors. Protein concentration was detected using a BCA test kit. The equal-volume proteins were separated by 12% SDS-PAGE, transferred to PVDF membrane, blocked with 5% skim milk for 2h, and rinsed with TBST 3 times (10 min/time). Then, NLRP3 (19771-1-AP, Proteintech, USA), Caspase-1 p20 (22915-1-AP, Proteintech), ASC (10500-1-AP, Proteintech), GSDMD-N (ab219800, Abcam, USA), p-p65 (3033, CST), GAPDH (2118, CST), and SPP1 (sc-21742, Santa Cruz Biotechnology) were separately added to incubate overnight at 4°C. Then, the HRP conjugate secondary antibody was supplemented for 1h before visualization of protein bands based on an enhanced chemiluminescence kit (Vazyme, China).

Wu X., Xin R., Zhang Y., Yang C., Sun F., Wang Y, & Zheng F. (2024). Xuebijing improves inflammation and pyroptosis of acute lung injury by up-regulating miR-181d-5p-mediated SPP1 inactivation. Clinics, 79, 100336.

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Detecting Protein Expression in Lung Tissue

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For detection of protein expression in lung tissue, samples were lysed in a 1% SDS buffer (1% SDS, 50mM triethanolamine pH 7.4, 150mM NaCl) containing a cocktail of phosphatase protease inhibitors (Sigma, 4906845001) and protease inhibitors (Thermo Scientific, A32965). The lysates were centrifuged at 15,000 rpm for 10 min and soluble protein supernatents were used for Western blot analysis. Equal amounts of protein (30ug) were separated by SDS-PAGE and transferred onto membranes. Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against GSDMD (abcam, ab219800) and actin (abcam, ab3280). For Western blotting of THP-1 cells, samples were lysed in 1% SDS containing protease inhibitors prior to SDS-PAGE separation as described above. Membranes were probed with antibodies against influenza virus nucleoprotein (abcam, ab20343), GSDMD (abcam, ab210070), and GAPDH (Thermo Scientific, ZG003).

Speaks S., Zani A., Solstad A., Kenney A., McFadden M.I., Zhang L., Eddy A.C., Amer A.O., Robinson R., Cai C., Ma J., Hemann E.A., Forero A, & Yount J.S. (2023). Gasdermin D promotes influenza virus-induced mortality through neutrophil amplification of inflammation. bioRxiv.

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Western Blot Analysis of Inflammasome Proteins

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Cells were prepared at 4°C in RIPA buffer (P0013J, Beyotime, China) containing proteinase inhibitor cocktail (B14001, Bimake, Houston, TX, USA) and phosphatase inhibitor cocktail (B15001, Bimake). Proteins were separated on 4%–20% precast mini polyacrylamide gels (SurePAGE™, GenScript, Nanjing, China) and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with anti-NEK7 (ab95873, Abcam), anti-GSDMD (ab219800, Abcam), anti-cleaved N-terminal GSDMD ab215203, Abcam), anti-NLRP3 (ab263899, Abcam), anti-cleavage caspase-1 (4199S, Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (9102S, Cell Signaling Technology), anti-p-ERK1/2 (9101S, Cell Signaling Technology), anti-α-SMA (A17910, ABclonal, Woburn, MA, USA), and anti-β-actin (66009-1-Ig, Proteintech, Rosemont, IL, USA). Membranes were then probed with appropriate secondary antibodies (Cell Signaling Technology). Immunoblot signals were detected by a Millipore chemical developer (Millipore Sigma, Burlington, MA, USA).

Yan Z., Da Q., Li Z., Lin Q., Yi J., Su Y., Yu G., Ren Q., Liu X., Lin Z., Qu J., Yin W, & Liu J. (2022). Inhibition of NEK7 Suppressed Hepatocellular Carcinoma Progression by Mediating Cancer Cell Pyroptosis. Frontiers in Oncology, 12, 812655.

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Histological Analysis of Rat Kidneys

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Rat kidneys were routinely processed for formaldehyde fixation, embedded in paraffin and positioned to obtain complete cross sections of the kidneys at the level of the renal papilla. Sections were first cut to a thickness of 4 μm and stained with haematoxylin and eosin (HE) for routine histological examination. Von Kossa staining was performed to visualize the crystals. Subsequently, the samples were sectioned for immunostaining with specific antibodies (cleaved caspase‐1 [WL02996a; Wanleibio], GSDMD [ab219800; Abcam]) against the target proteins. The procedure was conducted using the streptavidin‐peroxidase complex. The positive areas were outlined and then evaluated by a pathologist blinded to the experimental conditions as described previously.³³

Liu J., Yang K., Jin Y., Liu Y., Chen Y., Zhang X., Yu S., Song E., Chen S., Zhang J., Jing G, & An R. (2020). H3 relaxin protects against calcium oxalate crystal‐induced renal inflammatory pyroptosis. Cell Proliferation, 53(10), e12902.

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Western Blot Analysis of Inflammasome Proteins

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Protein lysates of cell samples or NP tissues were prepared by RIPA lysis buffer plus 1% phenylmethanesulfonyl fluoride. The total protein concentrations were measured with a BCA protein assay kit (Santa Cruz). Proteins were then separated by 10% SDS‐PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were then blocked by 5% bovine serum albumin for 2 hours at room temperature, followed by incubation with primary antibodies at 4°C overnight. After that, the membranes were incubated with second antibodies (1:5000, Abcam, ab150077) at room temperature for 2 hours. Protein bands were then detected using an enhanced chemiluminescence kit (ThermoFisher) and quantified by Image J software. Antibodies against NLRP3 (1:2000, ab214185), IL‐18 (1:1000,ab71495), IL‐1β (1:2000, ab234437), Gasdermin D (GSDMD, 1:1000, ab219800), TSG101 (1:2000, ab125011), and GAPDH (1:5000, ab181602) were purchased from Abcam. Antibodies against CD9 (1:2000, #13403), CD81 (1:2000, #56039), CD63 (1:2000, #55051), GM130 (1:1000, #12480), Cleaved caspase‐1 (1:1000, #89332), cleaved GSDMD (1:1000, #50928) and Calnexin (1:1000, #2679) were purchased from CST.

Zhang J., Zhang J., Zhang Y., Liu W., Ni W., Huang X., Yuan J., Zhao B., Xiao H, & Xue F. (2020). Mesenchymal stem cells‐derived exosomes ameliorate intervertebral disc degeneration through inhibiting pyroptosis. Journal of Cellular and Molecular Medicine, 24(20), 11742-11754.

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Molecular Mechanisms of Estrogen-Mediated Autophagy

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Estradiol (E2758, purity ≥ 98%), MPP dihydrochloride hydrate (M7068, purity ≥ 97%), PHTPP (SML1355, purity ≥ 98%) and 3-Methyladenine (3-MA, M9281, purity ≥ 99%) were purchased from Sigma (Saint Louis, USA). Antibody for NLRP3 (19771-1-AP) and IL-18 (10663-1-AP) were purchased from Proteintech (Wuhan, China). Antibody for caspase-1 (48847) was purchased from SAB signalway antibody (Maryland, USA). Antibody for GSDMD (A18281) and IL-1β (A16288) were purchased from ABclonal (Wuhan, China). Antibody for Beclin 1 (ab210498), LC3B (ab192890), SQSTM1 (ab56416), Hcy (ab15154), estrogen receptor alpha (ERα) (ab32063), estrogen receptor beta (ERβ) (ab3576), GSDMD (ab219800), and β-actin (ab8226) were purchased from Abcam (Cambridge, UK). FITC - goat Anti-mouse IgG (ab6785), TRITC - goat anti-rabbit IgG (BS10250), FITC - goat anti-rabbit IgG (BS10950), HRP - goat anti-rabbit IgG (BS13278), and HRP - goat anti-mouse IgG (BS12478) were purchased from Bioworld (Bloomington, USA). The prestained protein MW marker (26617) were purchased from Thermo scientific. Control siRNA and ERα siRNA (sc-29305) were purchased from Santa Cruz (Santa Cruz, USA).

Meng Q., Li Y., Ji T., Chao Y., Li J., Fu Y., Wang S., Chen Q., Chen W., Huang F., Wang Y., Zhang Q., Wang X, & Bian H. (2020). Estrogen prevent atherosclerosis by attenuating endothelial cell pyroptosis via activation of estrogen receptor α-mediated autophagy. Journal of Advanced Research, 28, 149-164.

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