Sc 66846

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-66846 is a laboratory product offered by Santa Cruz Biotechnology. It is a piece of equipment designed for use in scientific research and analysis. The core function of Sc-66846 is to provide a specialized tool for researchers and laboratory technicians to aid in their work. No further details on the intended use or interpretation of this product are provided.

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Lab products found in correlation

Sc 33099, by Santa Cruz Biotechnology (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Sc 22514 r, by Santa Cruz Biotechnology (2 mentions) Bca assay, by Beyotime (2 mentions) Image lab 3, by Bio-Rad (2 mentions) Enhanced chemi luminescence (ecl), by Beyotime (2 mentions) Bs6007mh, by Bioworld Technology (2 mentions) Ab32047, by Abcam (2 mentions) Ab62352, by Abcam (2 mentions) Sc 2005, by Santa Cruz Biotechnology (2 mentions) Sc 1780, by Santa Cruz Biotechnology (2 mentions) Ripa assay, by Beyotime (2 mentions) X ray film, by Kodak (1 mentions) Asc sc 22514 r, by Santa Cruz Biotechnology (1 mentions) Caspase 1, by Cell Signaling Technology (1 mentions) Ab16911, by Abcam (1 mentions) Nitrocellulose membrane, by Merck Group (1 mentions) Sc 514, by Santa Cruz Biotechnology (1 mentions) Ecl western blotting detection system, by Merck Group (1 mentions) Ab18256, by Abcam (1 mentions)

Spelling variants of this product

NLRP3 (sc-66846 (2 citations)

10 protocols using sc 66846

Western Blot Analysis of Inflammatory Markers

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Western Blot studies were carried out as described previously (45 (link)–47 (link)). Briefly, control and experimental cells were harvested, lysed in RIPA buffer containing 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1mM EDTA, 1% NP-40, 0.25% Deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail (Calbiochem, Cocktail Set I), 1mM PMSF, and 0.2mM sodium orthovanadate. Protein concentration was determined using the Bio-Rad Protein Assay kit (PIERCE, Rockford, IL). Total protein lysed extracts (30 μg/lane) were loaded on a 10 % polyacrylamide (PAGE) premade gel (Bio-Rad, Hercules, CA) and after transferring onto PVDF membrane were processed for immunostaining with primary antibodies against APOL1 (anti-mouse, #66124-I-IG, Protein tech), NLRP3 (anti-mouse, #sc-66846; Santa Cruz); ASC (sc-22514-R; Santa Cruz Biotechnology), Caspase-1and Cleaved (C ) Caspase-1 (antirabbit #4199;1:700, Cell Signaling): followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies. The blots were developed using a chemiluminescence detection kit (PIERCE, Rockford, IL) and exposed to X-ray film (Eastman Kodak Co., Rochester, NY). Equal protein loading and the protein transfers were confirmed by immunoblotting for determination of GAPDH protein using a monoclonal GAPDH antibody (#SC-47724; 1:3000, Santa Cruz) performed on the same (stripped) western blots.

Jha A., Kumar V., Haque S., Ayasolla K., Saha S., Lan X., Malhotra A., Saleem M.A., Skorecki K, & Singhal P.C. (2019). Alterations in Plasma Membrane Ion Channel Structures Stimulate NLRP3 Inflammasomes Activation in APOL1 Risk Milieu. The FEBS journal, 287(10), 2000-2022.

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Histochemical and Ultrastructural Analysis of Liver Tissue

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Sections of formalin-fixed livers were stained with hematoxylin-eosin (HE), Oil Red O, Sirius Red, and immunohistochemical staining for F4/80 (ab16911, Abcam, UK), NLRP3 (sc-66846, Santa Cruz, USA) and TXNIP (sc-33099, Santa Cruz, USA). The quantitative immunohistochemical staining values (QISVs) were calculated as the integrated OD divided by the total area occupied by the brown cells in each slide. The ultrastructures of the liver tissue were observed using a transmission electron microscopy (TEM, Hitachi, Japan).

He K., Zhu X., Liu Y., Miao C., Wang T., Li P., Zhao L., Chen Y., Gong J., Cai C., Li J., Li S., Ruan X.Z, & Gong J. (2017). Inhibition of NLRP3 inflammasome by thioredoxin-interacting protein in mouse Kupffer cells as a regulatory mechanism for non-alcoholic fatty liver disease development. Oncotarget, 8(23), 37657-37672.

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Hippocampal Protein Isolation and Western Blot Analysis

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By using ice-cold RIPA buffer the total protein was isolated from hippocampal tissues. BCA Protein Assay Kit (Thermo Fisher Scientific, USA) was used to measure the protein concentrations. A nitrocellulose membrane (Millipore Co., USA) was used for immuno-blotting where the protein samples (30 μg) were separated using SDS–polyacrylamide gel electrophoresis and then transferred to. For 1 h the membrane was blocked with 5% skim milk in Trisbuffered saline (150 mM NaCl, 0.1% Tween 20, 20 mM Tris, pH 7.4). Then the proteins were detected by incubation with primary antibodies against HMGB1, RAGE at 4 °C overnight. Later the membrane was washed for 3 times in Tween 20 + PBS and was incubated by the horseradish peroxidase-conjugated secondary antibodies for 2 h. The immuno-blots were visualized by a Millipore ECL Western Blotting Detection System and the variables protein expression levels were normalized to those of β-actin.
The following antibodies were used: anti-β actin (1:2,000, sc-130657, Santa Cruz Biotechnology), anti-HMGB1 (1:1,000, ab18256; Abcam), anti-RAGE antibody (1:2,000, sc8230; Santa Cruz), anti-NLRP3 antibody (1:2,000, sc-66846, Santa Cruz Biotechnology), anti-ASC antibody (1:2,000, sc-22514-R, Santa Cruz Biotechnology), pro and cleaved Caspase1 antibody (1:2,000, sc-514, Santa Cruz Biotechnology) and pro and mature IL-1β (1:1,000, AF-401-NA, R&D Systems).

Hendawy N., Salaheldin T.H, & Abuelezz S.A. (2023). PCSK9 Inhibition Reduces Depressive like Behavior in CUMS-Exposed Rats: Highlights on HMGB1/RAGE/TLR4 Pathway, NLRP3 Inflammasome Complex and IDO-1. Journal of Neuroimmune Pharmacology, 18(1-2), 195-207.

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Proteolytic Cleavage of Inflammatory Mediators

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Recombinant human IL-1β, NLRP-3 or PYCARD (ASC) (280 ng, H00003553-P02, H00114548-P01 or H00029108-P01, Abnova) were incubated with recombinant active human MMP-7 (0.035U, #444270 Merck Millipore) in MMP reaction buffer (20 mM Tris, pH 7.6, 5 mM CaCl₂, 0.1 M NaCl) at 37°C until stopped with 100 mM DDT. Fragments were detected by Western blot, using rabbit anti-IL-1 beta (1:2 000, ab9722, Abcam), rabbit anti-ASC (1:1 000, p9522-75, US Biological) and rabbit anti-NLRP3 (1:500, sc-66846, Santa Cruz).

Ambite I., Puthia M., Nagy K., Cafaro C., Nadeem A., Butler D.S., Rydström G., Filenko N.A., Wullt B., Miethke T, & Svanborg C. (2016). Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets. PLoS Pathogens, 12(10), e1005848.

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Western Blot Analysis of Inflammatory Proteins

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Total protein was extracted in cell lysis buffer (R0278, RIPA buffer, Sigma, UK) and the protein concentration was determined. The protein concentration was determined with a BCA Protein Assay Kit (23227, Thermo, UK). Equal amounts of proteins per sample were separated by electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked for 1 h with 5% albumin bovine and incubated with primary antibodies specific for TXNIP (sc-33099, Santa Cruz, USA), NLRP3 (sc-66846, Santa Cruz, USA), ASC (sc-22514-R, Santa Cruz, USA) and Caspase-1 (sc-56036, Santa Cruz, USA) at 4°C overnight. The membranes were washed and incubated with the secondary antibodies. Protein expression was assessed by chemiluminescence. Relative amounts of protein were quantified based on the relative densities of the protein bands, by using an image analysis system (Bio-Rad Gel Doc 2000, USA).

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NLRP3 Protein Expression Quantification

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Proteins from lysates of BMDMs were extracted in Radio-Immunoprecipitation (RIPA) Buffer (Sigma Aldrich) containing Halt^TM protease and phosphatase inhibitor cocktail (Fisher Scientific, Napean, ON, Canada). Immunoblots were prepared with Bolt^® Bis-Tris Plus Gel (ThermoFisher), and western blot analysis was carried out according to standard protocols, with antibodies specific for rabbit polyclonal raised against human NLRP3 (1:6500, sc-66846, Santa Cruz Biotechnology Inc., Santa Cruz, CA). GAPDH was used as a loading control (AM4300, ThermoFisher). Relative protein levels were normalized to GAPDH as determined by densitometry using Image J software (1.8v, NIH).

Alphonse M.P., Duong T.T., Tam S, & Yeung R.S. (2023). Mercury increases IL-1β and IL-18 secretion and intensifies coronary arteritis in an animal model of Kawasaki disease. Frontiers in Immunology, 14, 1126154.

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Western Blot Analysis of Inflammatory Markers

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Colon tissue samples or cell samples or supernatant samples were split using RIPA assay (Beyotime) in ice. Total proteins were quantified using BCA assay (Beyotime) and were electrophoresed on 10% SDS-acrylamide gels. Total proteins were transferred to nitrocellulose membranes and membranes were blocked with 5% non-fat milk in TBS for 1 h at 37° C. Membranes were incubated with p-AMPK (ab23875, abcam), AMPK (ab32047, abcam), Nrf2 (ab62352, 1:1000, abcam), GSDMD (ab219800, 1:1000, abcam), NLRP3 (sc-66846, 1:500, Santa Cruz, USA), caspase-1 (sc-1780, 1:500, Santa Cruz, USA), and β-Actin (BS6007MH, 1:5000, Bioworld Technology, Inc.) at 4° C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:5000, Santa Cruz, USA) for 1 h at 37° C after washing with TBST for 15 min. Protein was measured using an enhanced chemiluminescence system (ECL, Beyotime) and analyzed using an Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Zhang W., Wang W., Shen C., Wang X., Pu Z, & Yin Q. (2021). Network pharmacology for systematic understanding of Schisandrin B reduces the epithelial cells injury of colitis through regulating pyroptosis by AMPK/Nrf2/NLRP3 inflammasome. Aging (Albany NY), 13(19), 23193-23209.

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Visualizing Caspase-1 Activation and NLRP3 Inflammasome Formation

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Caspase-1 activation and active caspase-1 foci formation was determined by FAM-YVAD-FMK (Immunochemistry Technologies) staining of macrophages infected on glass coverslips. DNA was stained with Hoescht 33342. Cells were washed with PBS and subsequently fixed with Cytofix (BD Biosciences). Immunofluorescent staining was performed post-fixation in PermWash (BD Biosciences) with antibodies specific to NLRP3 (sc-66846, Santa Cruz), ASC (ADI-905-173-100, Enzo), or pro-caspase-1 (sc-514, Santa Cruz) with Alexa fluor-conjugated secondary antibodies (Invitrogen). Coverslips were mounted with ProLong (Molecular Probes) and examined by confocal microscope (Leica SP8X) at the W. M. Keck Microscopy Center. Caspase-1 activation or foci formation was enumerated by counting the fraction of positive cells in four separate fields for each coverslip. Caspase-1 and ASC foci colocalization were enumerated by counting the fraction of positive cells in eight separate fields from duplicate coverslips.

Carpentier S.J., Ni M., Duggan J.M., James R.G., Cookson B.T, & Hamerman J.A. (2019). The signaling adaptor BCAP inhibits NLRP3 and NLRC4 inflammasome activation in macrophages through interactions with Flightless-1. Science signaling, 12(581), eaau0615.

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Multiplexed Immunostaining for SPHK, NLRP3, and GLUT1

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Primary antibodies included rabbit polyclonal to SPHK1 and SPHK2 (Bs-2652R and Bs-2653R, Bioss, Woburn, MA, USA, both 1:50), NLRP3 and IL-1β (sc-66,846 and sc-7884, Santa Cruz, Dallas, TX, USA, both 1:40), GLUT1 (Glucose Transporter 1, ab652, Abcam, Cambridge, UK, 1:100), goat polyclonal antibody to SPNS2 (sc-165,572, Santa Cruz, 1:40), TNFα (sc-1348, Santa Cruz, 1:50), myeloperoxidase (#2141, Osenses, Adelaide, SA, Australia, 1:100), and a mouse monoclonal antibody to neutrophil elastase (M0752, Dako GmbH, Jena, Germany, 1:100). Conjugated antibodies (1:150) were F (ab’)2 fragments of donkey IgG, obtained from Jackson ImmunoReseach (West Grove, PA, USA), anti-rabbit IgG (Alexa Fluor 594 or Alexa Fluor 488), anti-goat IgG (Alexa Fluor 488), and anti-mouse IgG (Alexa Fluor 647).

Tran H.B., Macowan M.G., Abdo A., Donnelley M., Parsons D, & Hodge S. (2020). Enhanced inflammasome activation and reduced sphingosine-1 phosphate S1P signalling in a respiratory mucoobstructive disease model. Journal of Inflammation (London, England), 17, 16.

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Western Blot Analysis of Oxidative Stress Markers

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Tissue samples or cell samples or supernatant samples were splitted using RIPA assay (Beyotime) in ice. Total proteins were quantified using BCA assay (Beyotime) and were electrophoresed on 10% SDS-acrylamide gels. Total proteins were transferred to nitrocellulose membranes, and membranes were blocked with 5% nonfat milk in TBS for 1 h at 37°C. Membranes were incubated with AdipoR1 (ab50675, 1 : 2000, abcam), AMPK (ab32047, 1 : 2000, abcam), p-AMPK (ab133448, 1 : 1000, abcam), TXNIP (ab188865, 1 : 2000, abcam), NRF2 (ab62352, 1 : 2000, abcam), HO-1 (ab52947, 1 : 2000, abcam), sOD2 (ab68155, 1 : 2000, abcam), GPX4 (ab41787, 1 : 2000, abcam), GSDMD (ab209845, 1 : 2000, abcam), NLRP3 (sc-66846, 1 : 500, Santa Cruz, USA), caspase-1 (sc-1780, 1 : 500, Santa Cruz, USA), IL-1β (sc-12742, 1 : 500, Santa Cruz, USA), and β-actin (BS6007MH, 1 : 5000, Bioworld Technology, Inc.) at 4°C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1 : 5000, Santa Cruz, USA) for 1 h at 37°C after washing with TBST for 15 min. Protein was measured using an enhanced chemiluminescence system (ECL, Beyotime) and analyzed using an Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Wang X., Li Q., Sui B., Xu M., Pu Z, & Qiu T. (2022). Schisandrin A from Schisandra chinensis Attenuates Ferroptosis and NLRP3 Inflammasome-Mediated Pyroptosis in Diabetic Nephropathy through Mitochondrial Damage by AdipoR1 Ubiquitination. Oxidative Medicine and Cellular Longevity, 2022, 5411462.

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