Gasdermin D was measured by an ELISA method as we previously described (Xiong et al., 2018 (link)). In short, 100 μL of serum samples or standard (recombinant Human GSDMD protein; Abcam, United States) were added into each capture antibody-coated well (anti-GSDMD antibody; Abcam, United States) and incubated for 1 h at 37°C. After aspirating and washing each well three times, 100 μL of diluted detection antibody-conjugated HRP (rabbit anti-human IgG H&G antibody; Abcam, United States) was added and incubated for 30 min at 37°C. Repeating the aspiration/wash of each well three times, 100 μL of substrate solution was added and incubated at room temperature for 30 min. Next, 50 μL of stop solution (1 mol/L H2SO4) was added to each well. The absorbance of the colored solution of GSDMD was measured at 450 nm by using a MultiskanTM FC microplate reader (Thermo Fisher Scientific, Waltham, MA, United States).
Anti gsdmd antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-GSDMD antibody is a laboratory reagent used for the detection and analysis of GSDMD (gasdermin D) protein. GSDMD is a key executor of pyroptosis, a form of programmed cell death. This antibody can be used in various immunological techniques, such as Western blotting, immunohistochemistry, and immunofluorescence, to investigate the expression and localization of GSDMD in biological samples.
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Lab products found in correlation
Anti pro caspase1 p10 p12 antibody, by Abcam (2 mentions)
Multiskan fc microplate reader, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Anti pcna antibody, by Abcam (1 mentions)
Anti h3 antibody, by Proteintech (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti 4 hne antibody, by Abcam (1 mentions)
Anti hdac3 antibody, by Proteintech (1 mentions)
Anti cox4 antibody, by Abcam (1 mentions)
Protein a g beads, by Merck Group (1 mentions)
Normal sheep serum, by ZSGB-BIO (1 mentions)
Gapdh mouse monoclonal antibody, by Proteintech (1 mentions)
Coralite594 conjugated donkey anti rabbit igg h l, by Proteintech (1 mentions)
Tunel apoptosis assay kit, by Keygen Biotech (1 mentions)
Triton x 100, by Keygen Biotech (1 mentions)
Cyclophosphamide, by Baxter (1 mentions)
Anti caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Tmr red kit, by Roche (1 mentions)
Cell lysis buffer 10, by Cell Signaling Technology (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti gsdmd antibody
1
Quantification of Gasdermin D by ELISA
2
Western Blot Analysis of Protein Acetylation
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Protein samples were separated by 8%–12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). After blocking with 5% milk in TBST for 1h, the membranes were incubated overnight at 4°C with primary antibodies. Subsequently, membranes were washed and incubated with secondary antibodies (1:10,000) and detected using the chemiluminescence method. The intensity of the bands was quantified by ImageJ software. Antibodies included anti-NLRP3 antibody (1:1,000, CST), anti-GSDMD antibody (1:1,000, Abcam), anti-N-GSDMD antibody (1:1,000, Abcam), anti-Capspase-1 p20 antibody (1:1,000, AdipoGen), anti-4-HNE antibody (1:1,000, Abcam), anti-HDAC3 antibody (1:1,000, Proteintech), anti-HADHA antibody (1:1,000, Abcam), anti-Ace-lys antibody (1:1,000, Abcam), anti-H3 antibody (1:1,000, Proteintech), anti-COX4 antibody (1:1,000, Abcam), anti-PCNA antibody (1:1,000, Abcam). Anti-GAPDH (1:5,000, Invitrogen) was used as an internal control.
For acetylation-immunoprecipitation, cells were lysed with immunoprecipitation buffer [supplemented with TSA (10 mM)] and sonicated. The samples were immunoprecipitated with protein A/G beads (Sigma) overnight at 4°C, washed three times in lysis buffer, resolved by loading buffer, and analyzed by Western blotting.
For acetylation-immunoprecipitation, cells were lysed with immunoprecipitation buffer [supplemented with TSA (10 mM)] and sonicated. The samples were immunoprecipitated with protein A/G beads (Sigma) overnight at 4°C, washed three times in lysis buffer, resolved by loading buffer, and analyzed by Western blotting.
3
Ovarian Dysfunction Mechanism Exploration
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The chemicals and reagents used were the standard substances kaempferol, quercetin, hesperetin, and hesperidin (National Institutes for Food and Drug Control), Cyclophosphamide (Baxter Oncology GmbH, Halle, Germany; No. 8K274A), Dehydroepiandrosterone (General Nutrition Corporation Pittsburgh, PA, USA; No. 61411H18), Rat FSH enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone Corp. Wuhan, China; No. CEA830Ra), Rat AMH ELISA kit (Cloud-Clone Corp. Wuhan, China; No. CEA228Ra), Rat GnRH ELISA kit (Cloud-Clone Corp. Wuhan, China; No. CEA843Ra), Rat E2 ELISA kit (Cloud-Clone Corp. Wuhan, China; No. CEA461Ge), TUNEL apoptosis assay kit (KeyGENBio TECH. Jiangsu, China; No. KGA 7072), 4′, 6-diamidino-2-phenylindole (DAPI) staining kit (KeyGENBio TECH. Jiangsu, China; No. KGA 215-10), Normal sheep serum (ZSGB-BIO. Beijing, China; No. ZLI-9022), Triton X-100 (KeyGENBio TECH. Jiangsu, China; No. KGF011), Caspase-1 rabbit polyclonal antibody (Proteintech Group, Inc. Chicago, USA; No. 22915-1-AP), GAPDH mouse monoclonal antibody (Proteintech Group, Inc. Chicago, USA; No. 60004-1-lg), Anti-GSDMD antibody (Abcam Cambridge, MA, USA; No. ab219800), Rat IL-18 ELISA kit (RayBiotech, Inc. Georgia, USA; No. P97636), and CoraLite594-conjugated donkey anti-rabbit IgG (H+L) (Proteintech Group, Inc. Chicago, USA; No. SA00013-8).
4
Immunoblotting Analysis of Inflammatory Pathways
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Anti-cleaved caspase-3 antibody, anti-caspase-3 antibody, anti-β-actin antibody, anti-RIP1 (receptor-interacting protein 1) antibody, anti-p-RIP1 (phospho-RIP1) antibody, cell lysis buffer (10×), and protease/phosphatase inhibitor cocktail were all from Cell Signaling Technology (Danvers, MA). Anti-caspase-11 antibody, anti-pro caspase-1+p10+p12 antibody, anti-GSDMD antibody, anti-GSDME antibody, and propidium iodide were from Abcam (Cambridge, UK). Anti-GFP antibody, Hoechst 33342, MitoTracker Deep Red FM, and LysoTracker Deep Red were from Invitrogen. Anti-F4/80 antibody was from BD Biosciences (Franklin Lakes, NJ). TMR red kit was from Roche (Basel, Switzerland). Lipofectamine 3000 Transfection Reagent was from Thermo Fisher Scientific.
5
GSDMD, Caspase-1, and IL-1β Detection
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Anti-GSDMD antibody, anti-pro-caspase-1 + p10 + p12 antibody, and anti-IL-1β antibody for western blotting were all purchased from Abcam (Cambridge, UK). Anti-NLRP3 antibody was purchased from Wanleibio (Shenyang, China). Anti-GSDMD antibody for immunofluorescence was purchased from Bioss (Beijing, China). MCC950 (NLRP3 inhibitor) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). rTFPI was purchased from R&D (Minneapolis, MN, USA).
6
Immunohistochemical Analysis of GSDMD, NLRP3, and NF-κB
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Immunohistochemistry was performed according to a previous description (Liu et al., 2016 (link)). A series of spinal cord tissue processing steps were conducted, such as perfusion, fixation, embedding, dewaxing, rehydration, deparaffinization, and antigen retrieval. At 4°C, the sections were then cultured with anti-GSDMD antibody (1:1000; Abcam), anti-Nlrp3 (1:1000; Proteintech), and anti-NF-κB/p65 antibody (1:500; Abcam) overnight. Thereafter, the incubation of the sections with a secondary antibody was maintained at 37°C for 30 min. After staining, an Olympus BX60 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) was applied to acquire images of the specimens.
7
Western Blot Analysis of GSDMD
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Homogenization of lung tissues was done at freezing temperature in RIPA buffer containing phosphatase and protease inhibitors. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate protein specimens, which were then loaded onto a PVDF membrane. After blocking the membrane with 5 percent skim milk, an Anti-GSDMD antibody (1 : 1000, ab, Abcam, UK) was applied, and the specimens were subjected to incubation at a temperature of 4°C over the night. The membranes were once again incubated for 2 hours at ambient temperature with rabbit HRP-conjugated secondary antibody and the chemiluminescent peroxidase substrate (Millipore, Boston, MA, USA) was utilized to visualize the protein bands. ImageJ software (National Institutes of Health, USA) was utilized to quantify the densitometric analysis.
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