Anti caspase 1 p20

Manufactured by Adipogen

Sourced in United States

Anti-caspase-1 p20 is a laboratory reagent used for the detection and quantification of the p20 subunit of caspase-1 enzyme. Caspase-1 is an important mediator of the inflammatory response and plays a key role in the activation of cytokines such as IL-1β and IL-18. The anti-caspase-1 p20 reagent can be used in various experimental techniques to study the activation and regulation of caspase-1.

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Lab products found in correlation

Anti nlrp3, by Adipogen (4 mentions) Anti asc, by Adipogen (3 mentions) Phopho akt ser473 c d9e, by Cell Signaling Technology (2 mentions) S 2 3 bis palmitoyloxy 2 rs propyl n palmitoyl r cys ser lys4 oh pam3cys, by InvivoGen (2 mentions) Mpges inhibitor cay10526, by Cayman Chemical (2 mentions) Pge2 ep4 antagonist bgc 20 1531 ep2 antagonist pf 04418948 ep3 antagonist l 798 106 akt1 2 inhibitor akti 1 2 pka inhibitor h89 dihydrochloride, by Bio-Techne (2 mentions) Epac inhibitor esi 09, by Bio-Techne (2 mentions) Polyinosinic polycytidylic acid poly 1 c, by InvivoGen (2 mentions) Anti p p38, by Cell Signaling Technology (2 mentions) Anti p jnk, by Cell Signaling Technology (2 mentions) Anti il 1β, by GeneTex (2 mentions) Anti il 1β, by R&D Systems (2 mentions) Anti β actin, by Merck Group (1 mentions) Bca protein assay kit, by Bio-Rad (1 mentions) Ecl western blot substrate, by Thermo Fisher Scientific (1 mentions) Pierce ip lysis buffer, by Thermo Fisher Scientific (1 mentions) Anti p65, by Cell Signaling Technology (1 mentions) Anti p p65 ser536, by Cell Signaling Technology (1 mentions) Anti cith3, by Abcam (1 mentions) Anti histone h2b, by Abcam (1 mentions)

Close spelling variants of this product

Caspase-1 (36 citations) Anti-caspase-1 (19 citations) Mouse anti-caspase 1 (p20) (12 citations) Caspase-1 p20 (10 citations) Anti-mouse caspase-1 p20 (AG-20B-0042) (6 citations) Anti-caspase-1 (P20) (AG-20B-0042-C100) (3 citations) Anti-caspase-1 (p20) mAb (2 citations)

18 protocols using anti caspase 1 p20

Lung Tissue Protein Analysis

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Lung tissue was lysed in Pierce™ IP Lysis Buffer (ThermoFisher Scientific). The lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected and the protein concentration in the supernatant was determined by BCA Protein Assay Kit (Bio-Rad Laboratories). For the immunoblot, 40 µg of the total protein was resolved in SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk in PBS containing 0.1% Tween-20 for 30 min at room temperature. The membrane was incubated with primary antibodies overnight at 4 °C followed by incubation with secondary antibodies. The membrane was then developed using ECL Western Blot Substrate (ThermoFisher Scientific). The following primary antibodies were used: anti-NLRP3 (1:1000, AG-20B-0014-C100), anti-Caspase-1(p20) (1:1000, AG-20B-0042-C100), anti-Asc (1:500, AG-25B-0006-C100) from Adipogen Life Sciences, Anti-p65 (8242, 1:1000), anti-P-p65-Ser536 (3033, 1:500) from Cell Signaling Technology Inc., and anti-β-actin (1:5000, A5441, Sigma-Aldrich). For the immunoblot analysis of BALF samples, equal volumes of BALF from all groups were loaded. The following primary antibodies were used: anti-Histone H2B (1:500, ab52484, Abcam), anti-CitH3 (1:500, ab5103, Abcam),

Nagre N., Nicholson G., Cong X., Lockett J., Pearson A.C., Chan V., Kim W.K., Vinod K.Y, & Catravas J.D. (2022). Activation of cannabinoid-2 receptor protects against Pseudomonas aeruginosa induced acute lung injury and inflammation. Respiratory Research, 23, 326.

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Immunoblotting and Pharmacological Inhibition

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Antibodies against cPLA2 (D49A7), phospho-cPLA2 (D4I2A), IRF3 (D83B9), phospho-IRF3(4D4G), phospho-TBK-1 (D52C2), phospho-JNK (81E11), phospho-ERK1/2 (D13.14.4E), p38 (D13E1), phospho-p38 (D3F9), IκBα (44D4 and 9242), Syk (2712), phospho-Syk TYR525/526 (C87C1), AKT (11E7), and phopho-AKT Ser473 c(D9E) were obtained from Cell Signaling. Anti-Caspase-1 p20 was obtained from Adipogen. Protein-free phenol/water-extracted Escherichia-coli K235 LPS was prepared as described previously ^{65 (link)}. S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys₄-OH (Pam3Cys) and polyinosinic:polycytidylic acid (poly I:C; VacciGrade) was purchased from InvivoGen. Purified Adenylate Cyclase Toxin (AC) from Bordetella pertussis was a gift of E. Hewlett (Univ. of Virginia) and has been described previously^{39 (link)}. The mPGES inhibitor (CAY10526) was obtained from Cayman Chemical. PGE₂, EP4 antagonist (BGC 20–1531), EP2 antagonist (PF-04418948), EP3 antagonist (L-798,106), AKT1&2 inhibitor (AKTi-1/2), PKA inhibitor (H89 dihydrochloride), and EPAC inhibitor (ESI 09) were obtained from Tocris Bioscience.

Perkins D.J., Richard K., Hansen A.M., Lai W., Nallar S., Koller B, & Vogel S.N. (2018). Autocrine-Paracrine Prostaglandin E2 Signaling Restricts TLR4 internalization and TRIF Signaling. Nature immunology, 19(12), 1309-1318.

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Immunoblotting and Pharmacological Inhibition

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Analysis of Macrophage Signaling Pathways

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Macrophage cell lysates were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktails and PhosSTOP (Roche). Protein concentration was measured using the BCA Protein Assay Kit (Pierce). Primary antibodies used were anti-caspase-1 p20 (AdipoGen, no. AG-20B-0042; 1:1000), LC-3 (Novus Biologicals, no. NB100-2220; 1:500), P62 (Novus Biologicals, no. NBP1-48320; 1:4000), NLRP3 (Adipogen, no. AG-20B-0014; 1:1000), ASC (Adipogen, no. AG-25B-0006; 1:1000), PDHe1α (ThermoFisher, no. PA5-21536; 1:1000), p-AMPK (Cell Signaling, no. 50081s; 1:1000), p-ACC (Cell Signaling, no. 3661s; 1:750), TUFM (Novus Biologicals, no. CL2242; 1:500), GAPDH (Invitrogen, no. MA5-15738; 1:10,000), OPA1 (Cell Signaling, no. CST80471; 1:1000), p-DRP1 (Ser616) (Cell Signaling, no. 3455S; 1:500), p-TBK1 (Ser172) ( Cell Signaling, no. CST5483; 1:1000); and β-actin (Sigma-Aldrich, no. A5441; 1:5000). Immunoblots were visualized with the Supersignal substrate system (Thermo Fisher Scientific), and chemiluminescence was captured using the ChemiDox MP imaging system (Bio-Rad) or an LSA-3000 imaging system (Fujifilm Life Science).

Meyers A.K., Wang Z., Han W., Zhao Q., Zabalawi M., Duan L., Liu J., Zhang Q., Manne R., Lorenzo F.R., Quinn M., Song Q., Fan D., Lin H., Furdui C.M., Locasale J.W., McCall C.E., & Zhu X. (2021). Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation.

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Inflammasome Activation Assay Protocol

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BMDCs (2 × 10⁶) were treated with or without blocking antibody for 60 min before the addition of live or heat-killed H. capsulatum. For inflammasome analysis, cells were cultured in medium containing 0.1% heat-inactivated FBS and for signaling molecule analysis, in medium containing 10% FBS. Cells were detached from the wells and lysed with PhosphoSafe lysis buffer (MERCK) at different time points. Harvested cell-free supernatants were concentrated by 10-fold with Vivaspin 500 (GE Healthcare). Cell lysates were subjected to electrophoresis at 10% (for cell lysates) or 12.5% (for supernatants) SDS polyacrylamide gel and transferred to a 0.45 (for cell lysates) or 0.22 (for supernatants) μm PVDF membrane. The membrane was blocked with 5% non-fat milk and left in buffer containing anti-IL-1β p17 (R&D system), anti-Caspase-1 p20 (Adipogen), anti-NLRP3 (Adipogen), anti-ASC (Adipogen), anti-p-Syk (Abcam), anti-p-ERK (Cell Signaling), anti-p-JNK (Cell Signaling), anti-p-p38 (Cell Signaling) or anti-β-actin (GeneTex) antibody at 4°C overnight. The membrane was washed with TBST before addition of HRP-conjugated anti-goat IgG (1:3000), anti-rabbit (1:20,000) or anti-rat IgG (1:20,000). ECL reagent (PerkinElmer Life Science, Merck Millipore and GE Healthcare) was used for detection.

Chang T.H., Huang J.H., Lin H.C., Chen W.Y., Lee Y.H., Hsu L.C., Netea M.G., Ting J.P, & Wu-Hsieh B.A. (2017). Dectin-2 is a primary receptor for NLRP3 inflammasome activation in dendritic cell response to Histoplasma capsulatum. PLoS Pathogens, 13(7), e1006485.

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Immunoblotting Analysis of Kidney and Podocyte Proteins

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For immunoblotting analysis, electrophoresis of kidney cell lysates or cell lysates from the NZM2328 podocyte cell line was conducted with 10% running gels and 5% stacking gels. Proteins from the cell lysates were then transferred onto a polyvinylidene fluoride membrane. After blockade with 5% non-fat dry milk in Tris buffered saline with 0.1% Tween 20 (TBST), membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-RIP3 Ab (Abcam), anti-p-RIP3 Ab (Abcam), anti-MLKL (Abcam), anti-p-MLKL Ab (Abcam), mouse anti-NLRP3 (Adipogen), anti-caspase-1 p20 (Adipogen), rabbit anti-caspase-8 (Abcam) and anti-GAPDH Ab (Cell Signaling Technology). After washing 3 times with TBST, membranes were incubated with their corresponding secondary antibodies. The signals on the membranes were detected by a chemiluminescence analysis kit (Millipore).

Guo C., Fu R., Zhou M., Wang S., Huang Y., Hu H., Zhao J., Gaskin F., Yang N, & Fu S.M. (2019). Pathogenesis of Lupus Nephritis: RIP3 Dependent Necroptosis and NLRP3 Inflammasome Activation. Journal of autoimmunity, 103, 102286.

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Detecting Pro-Caspase-1 and IL-1β by Western Blot

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For western blot, the collected cell supernatants were subjected to TCA precipitation to pellet proteins, whereas cell lysates were prepared in SDS sample buffer. Both pro-caspase-1 and caspase-1-p20 were detected using anti-caspase-1(p20) (Adipogen, Cat#AG-20B-0042) at 1:1000 dilution. Pro-IL-1β and IL-1β (p17) were detected using anti-IL-1β (GeneTex Cat#GTX74034) at 1:1000 dilution. Blots were imaged using the BIO-RAD ChemiDoc MP imaging system as well as the Azure imaging system.

Strauch E., Kaspar H., Schaudinn C., Dersch P., Madela K., Gewinner C., Hertwig S., Wecke J, & Appel B. (2001). Characterization of Enterocoliticin, a Phage Tail-Like Bacteriocin, and Its Effect on Pathogenic Yersinia enterocolitica Strains. Applied and Environmental Microbiology, 67(12), 5634-5642.

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Inflammasome Protein Expression Analysis

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Anti-IL-1β, anti-apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and anti-cluster of differentiation (CD) 206 polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caspase-1 p-20 and NOD-like receptor protein 3 (NLRP3) monoclonal antibodies were obtained from Adipogen Life Sciences (San Diego, CA, USA). An anti-β-actin monoclonal antibody was obtained from Sigma–Aldrich. In some experiments, 5 mM ATP (Sigma–Aldrich) was applied to LPS-treated THP-1 cells for 30 min before collecting samples.

Kaneko J., Okinaga T., Hikiji H., Ariyoshi W., Yoshiga D., Habu M., Tominaga K, & Nishihara T. (2018). Zoledronic acid exacerbates inflammation through M1 macrophage polarization. Inflammation and Regeneration, 38, 16.

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Caspase-1 Activation in Monocytes

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To measure spontaneous or LPS-induced activation and secretion of caspase-1 in supernatants of monocytes, day 4 BM-derived cells (2 × 10⁶) were seeded in a 12-well plate in 1 ml of medium (DMEM containing 10% FCS). Cells were then left untreated for 4 h, washed with serum-free medium (DMEM), and incubated for 44 h or stimulated with 1 µg/ml LPS for 4 h, washed with DMEM, and incubated with or without 10 µM nigericin (Enzo Life Sciences) in DMEM for 44 h. Additionally, wild-type and Wdr1^rd/rd cells were pretreated with 1 µM latrunculin-b (Enzo Life Sciences) or 1 µM colchicine (Sigma-Aldrich) for 30 min before LPS priming for 4 h, washed with DMEM, and incubated for 44 h with or without latrunculin-b or colchicine. Culture supernatants were then collected and enriched by methanol chloroform precipitation (Masters et al., 2010 (link)). Precipitates were boiled for 5 min with SDS sample buffer, resolved by SDS/PAGE, and transferred to polyvinylidene fluoride membranes. After blocking in PBS with 0.1% Tween 20 and 5% skim milk for 1 h, membranes were then probed overnight at 4°C with anti–caspase-1 (p20; AdipoGen).

Kim M.L., Chae J.J., Park Y.H., De Nardo D., Stirzaker R.A., Ko H.J., Tye H., Cengia L., DiRago L., Metcalf D., Roberts A.W., Kastner D.L., Lew A.M., Lyras D., Kile B.T., Croker B.A, & Masters S.L. (2015). Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1β. The Journal of Experimental Medicine, 212(6), 927-938.

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Immunoblot detection of caspase-1 and tissue factor

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For detection of active caspase-1 and TF by Immunoblot, cells were washed with cold PBS and lysed with SDS sample buffer. Culture supernatants were precipitated with 1/10 volume of 2% sodium cholate and 1/10 volume of 100% trichloroacetic acid (TCA), and then dissolved in SDS sample buffer. Total protein from lysates and supernatants (equivalent to 5 ×10⁴ cells) was analyzed by fluorescent Immunoblot for multiplex detection. TF was detected using anti-tissue factor (Abcam, Cat#ab151748, rabbit monoclonal) at 1:1000 dilution. Both pro-caspase-1 and p20 caspase-1 were determined using anti-caspase-1(p20) (Adipogen, Cat#AG-20B-0042-C100 for mouse BMDMs, AG-20B-0048-C100 for THP-1 cells) at 1:1000 dilution. IL-1β (p17) was detected using anti-IL-1β (GeneTex Cat#GTX74034). Tissue factor and caspase-1 were visualized on the same blot in the 800 nm (IRDye 800CW) and 700 nm (IRDye 680RD) channel, respectively, with LI-COR Odyssey Classic Imager. Plasma fibrinogen (equivalent to 0.03 μl of plasma) was detected using anti-fibrinogen (Dako Cat#A0080, rabbit polyclonal) at 1:3000 dilution, and imaged in the 800 nm (IRDye 800CW) channel. Tissue fibrin was detected using anti-fibrin (59D8) at 1 μg/ml and imaged in the 700 nm (IRDye 680RD) channel.

Wu C., Lu W., Zhang Y., Zhang G., Shi X., Hisada Y., Grover S.P., Zhang X., Li L., Xiang B., Shi J., Li X.A., Daugherty A., Smyth S.S., Kirchhofer D., Shiroishi T., Shao F., Mackman N., Wei Y, & Li Z. (2019). Inflammasome activation triggers blood clotting and host death through pyroptosis. Immunity, 50(6), 1401-1411.e4.

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