Ag 25b 0006

Manufactured by Adipogen

Sourced in United States

The AG-25B-0006 is a laboratory equipment product. It serves a core function, but a detailed unbiased description cannot be provided while maintaining the requested conciseness and objectivity.

Automatically generated - may contain errors

Lab products found in correlation

Ag 20b 0014, by Adipogen (4 mentions) Gtx74034, by GeneTex (3 mentions) Ag 20b 0042 c100, by Adipogen (3 mentions) Ag 20b 0042, by Adipogen (3 mentions) Ag 20b 0014 c100, by Adipogen (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Lsm 510, by Zeiss (2 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions) Chemidoc mp, by Bio-Rad (1 mentions) Bca protein kit, by Beyotime (1 mentions) Af 401, by R&D Systems (1 mentions) Anti β actin hrp sc 47778, by Santa Cruz Biotechnology (1 mentions) Anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions) A3854, by Merck Group (1 mentions) Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions) Anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions) Sc 166692, by Santa Cruz Biotechnology (1 mentions) Tlrl 3pelps, by InvivoGen (1 mentions) Mlb00c, by R&D Systems (1 mentions) A5441, by R&D Systems (1 mentions)

Close spelling variants of this product

AG-25B-0006-C100 Anti-ASC (AG-25B-0006-C100, Anti-ASC (AG-25B-0006) ASC (AG-25B-0006)

19 protocols using ag 25b 0006

Western Blot Analysis of NLRP3 Inflammasome

Check if the same lab product or an alternative is used in the 5 most similar protocols

Kidneys harvested at the indicated times were homogenized in lysis buffer with protease inhibitors (Roche 4,693,159,001 and Roche 4,906,845,001). Total protein, measured with a bicinchoninic acid (BCA) protein kit (Beyotime Biotechnology Inc., China), was resuspended at 5 mg/ml. Total protein samples of the same volume and concentration were electrophoresed in SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Antibodies against NLRP3 (CST 15,101), Caspase 1 (Huaan ET1608-69), ASC (AdipoGen AG-25B-0006) and GAPDH (Zhongshan TA-08) were used after the membranes were blocked with 5% nonfat milk in Tris-buffered saline–Tween 20 (TBST) buffer, followed by incubation with goat anti-rabbit IgG H&L and goat anti-mouse IgG H&L (Zhongshan Corp., Beijing, China). The bands were visualized with a Bio-Rad ChemiDoc™ MP, and the intensity of the images was quantified using ImageJ 6.0 (National Institutes of Health, Bethesda, MD, USA).

Tan J., Dong L., Jiang Z., Tan L., Luo X., Pei G., Qin A., Zhong Z., Liu X., Tang Y, & Qin W. (2022). Probiotics ameliorate IgA nephropathy by improving gut dysbiosis and blunting NLRP3 signaling. Journal of Translational Medicine, 20, 382.

+ Open protocol

+ Expand

Immunoblotting Antibodies for Inflammation

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies for immunoblotting were used. Anti-β-actin-HRP (sc-47778, Santa Cruz Biotechnology or A3854, Sigma-Aldrich), goat anti-mouse IL-1β (AF-401, R&D systems), anti-mouse caspase-1 (Casper 1, AG-20B-0042-C100, Adipogen), anti-ASC (AG-25B-0006, Adipogen) and anti-NLRP3 (AG-20B-0014, Adipogen). Secondary anti-mouse and anti-goat antibodies were obtained from Santa Cruz Biotechnology, anti-rabbit secondary antibody was from Thermo Fisher Scientific.

Subbarao S., Sanchez-Garrido J., Krishnan N., Shenoy A.R, & Robertson B.D. (2020). Genetic and pharmacological inhibition of inflammasomes reduces the survival of Mycobacterium tuberculosis strains in macrophages. Scientific Reports, 10, 3709.

+ Open protocol

+ Expand

Western Blot Analysis of Inflammasome Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell lysates and culture supernatants were incubated with cell lysis buffer (20 mM Tris HCl pH 7.4, 200 mM NaCl, 1% NP-40) and denatured in laemmli buffer. Subsequently the protein samples were boiled at 95°C for 10 min and separated by SDS-PAGE. Separated proteins were transferred to PVDF membranes. Blocking, incubation with antibody and washing of the membrane were done in PBS supplemented with 0.05% Tween-20 (v/v) and 3% (w/v) non-fat dry milk. Immunoblots were incubated overnight with primary antibodies against caspase-1 (AG-20B-0042-C100, Adipogen), Nlrp3 (AG-20B-0014-C100, Adipogen), ASC (AG-25B-0006, Adipogen), IL-1β (GTX74034, Genetex), IL-18 (5180R-100, Biovision), IkBα (9242S, Cell Signaling), Phospho-IkBα (2859S, Ser32) (Cell Signaling), β-actin (NB600-501H, Novus Biologicals) and A20 (sc-166692, Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-mouse (115-035-146, Jackson Immunoresearch Laboratories) or anti-rabbit secondary antibody (111-035-144, Jackson Immunoresearch Laboratories) was used to detect proteins by enhanced chemiluminescence (Thermo Scientific).

Walle L.V., Van Opdenbosch N., Jacques P., Fossoul A., Verheugen E., Vogel P., Beyaert R., Elewaut D., Kanneganti T.D., van Loo G, & Lamkanfi M. (2014). Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis. Nature, 512(7512), 69-73.

+ Open protocol

+ Expand

Murine Airway Inflammation and Immune Response

Check if the same lab product or an alternative is used in the 5 most similar protocols

LPS (InvivoGen; tlrl-3pelps), alum (InvivoGen; tlrl-alk), recombinant murine IL-5 (PEPROTECH; 210-13), recombinant murine IL-1β (R&D Systems; 401-ML), SecinH3 (Abcam; ab145048), Brefeldin A (Wako; 022-15991), Cytochalasin B (Wako; 14930-96-2), OVA (MilliporeSigma; A2512-1G), HDM (Dermatophagoides pteronyssinus; LSL; LG-5449), and aluminium hydroxide gel (Wako; 012-24241) were purchased. Cytokines were measured by ELISA with mouse IL-1β (R&D Systems; MLB00C), mouse IL-5 (R&D Systems; M5000), mouse IL-13 (R&D Systems; M1300CB), mouse OVA-specific IgG1 antibody (Cayman Chemical; 500830), and mouse OVA-specific IgE antibody (BioLegend; 439807) kits according to the manufacturer’s protocols. Mouse monoclonal antibodies against β-actin (MilliporeSigma; A5441), caspase-1 (R&D Systems; MAB6215), IL-1β (Cell Signaling Technology; 3A6), and a rabbit polyclonal antibody against ASC (AdipoGen; AG-25B-0006) were purchased. Primary airway macrophages were isolated from BALF and were grown in RPMI 1640 with 10% FBS, 65 μg/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml fungizone.

Lee S., Ishitsuka A., Kuroki T., Lin Y.H., Shibuya A., Hongu T., Funakoshi Y., Kanaho Y., Nagata K, & Kawaguchi A. (2021). Arf6 exacerbates allergic asthma through cell-to-cell transmission of ASC inflammasomes. JCI Insight, 6(16), e139190.

+ Open protocol

+ Expand

SARS-CoV-2 Infection and Lung Pathology

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Tissues were obtained from autopsy of 11 patients with PCR confirmed SARS-CoV-2 and death due to COVID-related pneumonia and diffuse alveolar damage. Lung tissue from 3 patients with death due to non-pulmonary-related causes and no evidence of pneumonia or other pulmonary histopathological abnormalities were used as control. Tissues were fixed in 10% buffered formalin and paraffin embedded (FFPE). Immunofluorescence staining was performed on the Ventana Discovery Ultra instrument (Roche). Tissue sections were deparaffinized and antigen retrieval was performed using citrate buffer (pH = 8.0) at 72 ^oC (CC1; Roche). Tissue sections were first probed with rabbit anti-ASC (AG-25B-0006, AdipoGen) followed by anti-Rabbit HRP (760-4311, Roche) and fluorescent visualization using tyramide signal amplification (Red610, Roche). Residual peroxidase was blocked (760-4840, Roche), followed by application of mouse anti-CD68 (KP-1, Roche), OmniMap anti-mouse HRP (Roche) and visualization using Discovery Rhod6G (Roche). Representative immunofluorescence images were acquired using a Leica DMi8 microscope and for analysis the slides were scanned using a Zeiss AxiosScan Z1. Staining and imaging were performed by the Cedars-Sinai Biobank and Translational Research Core. ASC speck quantification was performed using open source QuPath software (version 0.2.3) (Bankhead et al., 2017 (link)).

Xian H., Liu Y., Rundberg Nilsson A., Gatchalian R., Crother T.R., Tourtellotte W.G., Zhang Y., Aleman-Muench G.R., Lewis G., Chen W., Kang S., Luevanos M., Trudler D., Lipton S.A., Soroosh P., Teijaro J., de la Torre J.C., Arditi M., Karin M, & Sanchez-Lopez E. (2021). Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation. Immunity, 54(7), 1463-1477.e11.

+ Open protocol

+ Expand

Visualizing Inflammasomes and Membrane Markers in BMDMs

Check if the same lab product or an alternative is used in the 5 most similar protocols

For visualization of inflammasomes, untreated or treated BMDMs were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 10% normal goat serum (005000121, Jackson ImmunoResearch) supplemented with 0.1% saponin (47036, Sigma) for 1 hr. Cells were incubated with a rabbit anti-ASC antibody (1:500 dilution, clone AL177, AG-25B-0006-C100, AdipoGen) overnight at 4 °C. An anti-rabbit secondary Rhodamine red antibody (111295144, Jackson ImmunoResearch) was used. Cells were counterstained in DAPI mounting medium (H-1200, Vecta Labs). Inflammasome specks and BMDMs were visualized, counted, and imaged using a Leica SP5 confocal microscope. For visualization of HBL and cell membrane, BMDMs were stimulated with HBL component B (5 μg/ml) for 1 hr, washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 1% BSA in PBS for 1 hr. Cells were incubated with a mouse anti-HBL-B antibody (1:200 dilution in 1% BSA)⁴⁰ and a rat FITC-conjugated anti-CD11b antibody (1:200 dilution in 1% BSA, 101205, BioLegend) overnight at 4 °C. PBS containing 0.05% Tween-20 was used to wash between incubation steps. An anti-mouse secondary Rhodamine red antibody (115295146, Jackson ImmunoResearch) was used. BMDMs were analysed using a Leica SP5 confocal microscope.

Mathur A., Feng S., Hayward J.A., Ngo C., Fox D., Atmosukarto I.I., Price J.D., Schauer K., Märtlbauer E., Robertson A.A., Burgio G., Fox E.M., Leppla S.H., Kaakoush N.O, & Man S.M. (2018). A multi-component toxin from Bacillus cereus incites inflammation and shapes host outcome via the NLRP3 inflammasome. Nature microbiology, 4(2), 362-374.

+ Open protocol

+ Expand

Microglia ASC Specks and Astrocyte NFκB

Check if the same lab product or an alternative is used in the 5 most similar protocols

The number of ASC specks within microglia and the subcellular localisation of NFκB within astrocytes were assessed by immunocytochemistry in cells seeded in 96-well plates. After treatment, cells were washed with DPBS (Gibco) and fixed in 4 % (v/v) PFA for 20 min at RT prior to further washing. Cells were permeabilised and non-specific binding blocked using 0.1 % (v/v) Triton-X-100, 5 % (w/v) BSA in TBS for 1 h at ambient temperature before incubating with an anti-ASC antibody (1:200, Adipogen, AG-25B-0006) or anti-NFκB (p65 subunit, 1:400, Cell signalling, D14E12) overnight at 4 °C. AlexaFluor 488 conjugated secondary antibody (Invitrogen) was added for 1 h at RT. Nuclei were stained with Hoechst-33342. Cells were imaged using a confocal Opera Phenix microscope (Perkin Elmer). The number of ASC specks per well was normalised to the number of nuclei and the mean nuclear intensity of NFκB was normalised to the total cell intensity per well using Harmony High-Content imaging analysis software (Perkin Elmer).

Beltran-Lobo P., Hughes M.M., Troakes C., Croft C.L., Rupawala H., Jutzi D., Ruepp M.D., Jimenez-Sanchez M., Perkinton M.S., Kassiou M., Golde T.E., Hanger D.P., Verkhratsky A., Perez-Nievas B.G, & Noble W. (2023). P2X7R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes. Brain, Behavior, and Immunity, 114, 414-429.

+ Open protocol

+ Expand

Immunoblotting Assay for B16F10 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For immunoblotting, B16F10 cells were lysed by PRO-PREP protein extraction solution (iNtRON Biotechnology). Briefly, 5 × 10⁶ cells were immersed in 400 μl of the PRO-PREP solution and homogenized in ice for 10–20 min. The mixture of cell lysates was then centrifuged at 13,000 rpm for 15 min. The supernatant was collected, and the protein concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The polypeptides were separated on 4–20% SDS–PAGE gradient gels and then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Ras Laboratories). The member was blocked with 5% skim milk (Difco Laboratories) and underwent overnight antibody incubation Primary Abs against the following proteins were used: human/mouse NLRP3 (1:1000, AP-20B-0014, AdipoGen), human NCOA6 (1:1000, A300-411A, Bethyl Laboratories), human/mouse ASC (1:1000, sc-514414, Santa Cruz Biotechnology; AG-25B-0006, AdipoGen), IL-1β (1:500, AF-401-NA, R&D Systems), mouse cleaved caspase-1 (1:1000, AG-20B-0042, AdipoGen), FLAG (1:1000, sc-166355, Santa Cruz Biotechnology), Myc (1:1000, ab13836, Abcam), GAPDH (1:1000, sc-25778, Sigma).

Li D., Wang X., Park D.J., Lee D.H, & Oh S. (2023). Inhibitory Effects of Latilactobacillus curvatus BYB3 Cell-Free Extract on Human Melanoma B16F10 Cells and Tumorigenic Mice. Journal of Microbiology and Biotechnology, 34(3), 589-595.

+ Open protocol

+ Expand

Western Blot Analysis of Inflammasome Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell lysates, serum samples and spleen homogenates denatured in loading buffer containing SDS and 100 mM DTT were boiled for 10 min before SDS–PAGE-separated proteins were transferred to PVDF membranes. Immunoblotting with primary antibodies against caspase-1 (1/3,000, kind gift of Dr Peter Vandenabeele, Ghent University), ASC (1/1,000, AG-25B-0006, Adipogen), IL-1β (1/2,000, GTX74034, GeneTex), caspase-11 (1/1,000, NB120-10454, Novus Biologicals), Ub-HRP (1/1,000, sc-8017, Santa Cruz), HMGB1 (1/100, 18256, Abcam), and β-actin (1/20,000, NB600-501H, Novus Biologicals) was followed by secondary anti-rabbit and anti-rat HRP antibodies (1/5,000, 111-035-144 and 112-035-143, Jackson Immunoresearch). Ponceau S Solution was purchased from Sigma-Aldrich.

Van Opdenbosch N., Gurung P., Vande Walle L., Fossoul A., Kanneganti T.D, & Lamkanfi M. (2014). Activation of the NLRP1b inflammasome independently of ASC-mediated caspase-1 autoproteolysis and speck formation. Nature Communications, 5, 3209.

+ Open protocol

+ Expand

Immunoblot Analysis of Inflammasome Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Lysis buffer containing 10 mM Tris-buffer (pH 7.6), 1% Triton X-100, 1% phosphatase inhibitor cocktail, and 1 mM PMSF was used to lyse cells. Cell lysates were then boiled in SDS sample buffer and resolved on a 10% SDS-PAGE gel. Immunoblots were incubated overnight with primary antibodies against NLRP3 (AG-20B-0014-C100, Adipogen, CH), ASC (AG-25B-0006, Adipogen, CH), mouse caspase-1 (AG-20B-0042-C100, Adipogen, CH), human caspase-1 (AG-20B-0048-C100, Adipogen, CH), IkBa (4814, Cell Signaling, USA), phospho-IkBa (2859, Ser32) (Cell Signaling, USA), A20 (23456-1-AP, Proteintech, USA), IL-1β (12242, Cell Signaling, USA) human GSDMD (sc-81868, Santa Cruz Biotechnology, USA), mouse GSDMD (ab209845, Abcam, USA), and GAPDH (10494-1-AP, Proteintech, USA). Immunoblots were examined using an ECL detection reagent (Millipore Corporation, Billerica, MA, USA).

Xue Z., Xi Q., Liu H., Guo X., Zhang J., Zhang Z., Li Y., Yang G., Zhou D., Yang H., Zhang L., Zhang Q., Gu C., Yang J., Da Y., Yao Z., Duo S, & Zhang R. (2019). miR-21 promotes NLRP3 inflammasome activation to mediate pyroptosis and endotoxic shock. Cell Death & Disease, 10(6), 461.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!