Proteins were separated from cells or tissues using RIPA buffer (EMD Millipore, Billerica, MA, USA). Then, protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher). The same concentration of samples was separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio–Rad Laboratories Inc., Hercules, CA, USA). Next, membranes were blocked with 5% non-fat dry milk at room temperature for 2 h. Then, they were incubated at 4℃ for 24 h with the following primary antibodies: anti-NLRP3 (1:500, Abcam, Cambridge, MA, USA), anti-pro-caspase-1 (1:1,000, Abcam), anti-cleaved-caspase-1 (1:500, Adipogen, San Diego, California, USA), anti-GSDMD-FL (1:500, Abcam), anti-GSDMD-N (1:500, Abcam), anti-Ngb (1:500, Abcam), and anti-GAPDH (1:1,000, Abcam). At room temperature, the membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. An enhanced chemiluminescence (ECL) kit (Bio–Rad, USA) was used to image the protein bands.
Anti gsdmd n
Manufactured by Abcam
Sourced in United States
Anti-GSDMD-N is a primary antibody that recognizes the N-terminal domain of the Gasdermin D (GSDMD) protein. GSDMD is a key executor of pyroptosis, a form of programmed cell death. This antibody can be used for the detection of GSDMD in various applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti gapdh, by Abcam (2 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Anti cleaved caspase 1, by Adipogen (1 mentions)
Anti pro caspase 1, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Anti nlrp3, by Abcam (1 mentions)
Rabbit anti il 1β, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Rabbit anti caspase 1, by Abcam (1 mentions)
Rabbit anti gsdmd, by Abcam (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Anti nlrp1, by Cell Signaling Technology (1 mentions)
Anti nlrp3, by Cell Signaling Technology (1 mentions)
Anti caspase 1, by Cell Signaling Technology (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Anti ha, by Novus Biologicals (1 mentions)
5 protocols using anti gsdmd n
1
Western Blot Analysis of Inflammasome Proteins
2
Hypoxia-Induced Inflammasome Activation
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Zinc nitrate hexahydrate (Zn(NO3)2·6H2O), 1,2-dimethylimidazole (1,2-MIL), and 1,3-diphenylisobenzofuran (DPBF) were purchased from Aladdin (China). TPZ was purchased from MCE (United States), while Ce6 was purchased from Frontier Scientific (United States). The primary antibodies against GAPDH, ASC, and NLRP3 were purchased from Proteintech (China). Anti-cleaved-caspase-1 and anti-hypoxia-inducible factor 1-alpha (HIF-1α) were purchased from CST (United States). Anti-GSDMD-N was purchased from Abcam (United States).
3
Western Blot Analysis of Neonatal Rat Brain
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Neonatal rats were deeply anesthetized with anhydrous 2-butanol and then sacrificed. Cortical tissues of the neonatal rat brain and HAPI microglial cells were lysed into homogenate in lysis buffer, and the extracted proteins (20 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA). Membranes were incubated with mouse anti-TIGAR (1:1000; Cat# sc-166290; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Caspase-1 (1:1000; Cat# ab207802; Abcam, Cambridge, UK), rabbit anti-GSDMD (1:1000; Cat# ab219800; Abcam), anti-GSDMD-N (1:1000; Cat# ab215203; Abcam), rabbit anti-IL-1β (1:1000; Cat# ab9787; Abcam), or mouse anti-β-actin (1:10 000; Cat# ab008; MULTI SCIENCES, Hangzhou, China) overnight at 4°C. The membranes were then incubated with peroxidase-conjugated rabbit/mouse secondary antibodies (1:5000; Cat# 111-035-003/115-035-003; Jackson, Lancaster, PA, USA), and bands were detected with an AmerSham Imager 600 (GE, Boston, MA, USA) and analyzed using ImageJ analysis software (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Quantification of Inflammatory Proteins
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Total protein was extracted from mouse kidney tissue and HUVECs using RIPA lysis buffer (Beyotime, cat. no. P0013B) on ice; the obtained lysate was centrifuged at 12,000 × g at 4°C for 30 min. Protein quantification was performed by using a BCA Protein Assay Kit (Thermo Fisher, cat.no. A53225). Proteins were separated by 10% SDS‐PAGE and then transferred to an NC membrane (Pall Bio Trace, cat. no. 66485) using a Bio‐Rad Trans‐Blot Turbo (USA) system. After blocking with 5% fat‐free milk powder in TBST buffer, the membranes were incubated with the following primary antibodies overnight at 4°C: anti‐GAPDH (abcam, cat. no. ab9485), anti‐caspase‐1 (Cell Signaling Technology, cat. no. 3866), anti‐NLRP3 (Cell Signaling Technology, cat. no. 13158), anti‐NLRP1 (Cell Signaling Technology, cat. no. 4990), anti‐GSDMD‐FL (Invitrogen, cat. no. PA5‐104324) and anti‐GSDMD‐N (abcam, cat. no. ab215203). Next, the membranes were incubated with horseradish peroxidase–conjugated anti‐rabbit IgG secondary antibodies for 2 h at room temperature. Bands were detected using a ChemiDoc MP Imaging System (Bio‐Rad), and ImageJ 1.51 software was used for the quantitative analysis of all protein bands.
5
Western Blotting of Protein Targets
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The liver tissues and cell lines were lysed using radioimmunoprecipitation assay (RIPA) buffer (#P1003B, Beyotime). Approximately 30 μg of total protein was added to 8% SDS PAGE gels using Bio-Rad equipment (Shanghai, China). After separation, the proteins were transferred to PVDF membranes (#ISEQ00010, Millipore, Massachusetts, USA). The protein-loaded PVDF membranes were blocked with 5% skim milk (#A600669, Sangon), immersed in solutions of the primary antibodies at 4 °C overnight, and then incubated with Alexa Fluor 680-conjugated secondary antibodies (Thermo Fisher Scientific). Tubulin-α or GAPDH was used as an internal control. The used primary antibodies were listed (anti-NLRP3, #IMG-6668A, Novus Biologicals, Colorado, USA; anti-MARCH7, #PA5-54572, Sigma Aldrich; anti-Cleaved caspase-1, #PA5-99390, Thermo Fisher Scientific; anti-Matured IL-1β, #AF401, R&D Systems, City of Emeryville, USA; anti-GSDMD-N, #GSDMD antibody Abcam EPR 19828, Abcam, Cambridge, UK; anti-Tubulin-α, #NB100-690, Novus Biologicals; anti-GAPDH, #MA1-16757, Thermo Fisher Scientific; anti-actin-β, #NB600-501, Novus Biologicals; anti-HA, #NB600-363, Novus Biologicals; anti-Flag, #MA1-91878, Thermo Fisher Scientific; anti-Ubiquitin, #NB300-130, Novus Biologicals; anti-GST, #13-6700, Thermo Fisher Scientific; anti-His, #NBP2-61482, Novus Biologicals; anti-Myc, #2276, Cell Signaling Technology, Boston, USA).
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