Sc 515

Manufactured by Santa Cruz Biotechnology

Sourced in United States

SC-515 is a laboratory filtration device designed for the separation and purification of biological samples. It utilizes a high-performance membrane to effectively filter and concentrate target analytes from complex matrices. The core function of SC-515 is to provide a reliable and efficient way to prepare samples for various analytical and research applications.

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Lab products found in correlation

Ab24170, by Abcam (1 mentions) Ab16667, by Abcam (1 mentions) Ag 20b 0014 c100, by Adipogen (1 mentions) Alexa fluor 633, by Thermo Fisher Scientific (1 mentions) Mab5326, by Merck Group (1 mentions) Z033429 2, by Agilent Technologies (1 mentions) Mab4401, by Merck Group (1 mentions) Ab5622, by Merck Group (1 mentions) Mms 435p, by Fortrea (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions) Ab9485, by Abcam (1 mentions) Ab195975, by Abcam (1 mentions) Proteinase and phosphatase inhibitor, by Roche (1 mentions) Anti pstat3 tyr705, by Cell Signaling Technology (1 mentions) Anti caspase 1, by Santa Cruz Biotechnology (1 mentions) Af410, by R&D Systems (1 mentions) Anti il 1β, by R&D Systems (1 mentions) Ecl substrate, by Thermo Fisher Scientific (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)

5 protocols using sc 515

Immunofluorescence Analysis of Neural Progenitor Cells

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Cultures were fixed in freshly prepared, buffered 4% paraformaldehyde. After blocking with 20% normal goat serum and permeabilization for 10min with 0.2% Triton X-100 in PBS, cultures were incubated overnight at 4°C with the following antibodies (mAb, monoclonal; pAb, polyclonal): Nestin ( MAB5326, Millipore, 1:200), Ki67 (ab16667, Abcam, 1:1000), Oct4 (MAB4401, Millipore, 1:2000), Pax6 (Developmental Studies Hybridoma Bank; 1:30), β-tubulin isotype III (β-tubIII, mAb, MMS-435P Covance, 1:400), Glial Fibrillary Acidic Protein (GFAP, z033429-2, Dako, 1:400), Lamp1 (pAb, ab24170, Abcam, 1:750), Cleaved Caspase-3 (pAb, #9661, Cell Signaling, 1:500), NLRP3 (pAb, AG-20B-0014-C100, AdipoGen, 1:200), Microtubule-Associated Protein 2 (MAP2, AB5622, Millipore, 1:400), Caspase 1 (Casp1, SC-515, Santa Cruz Biotechnology, 1:100), LC3B (pAb#2775, Cell Signaling, 1:400), GAPDH (ab9485, Abcam, 1:3000). After removal of the primary antibodies and repeated washes with PBS, cultures were incubated at room temperature for 45 min with secondary antibodies labeled with Alexa Fluor 633 or 488 (anti-mouse and/or anti-rabbit, Molecular Probes). Samples were then labeled with DAPI (0.3 μg/ml, Roche) for nuclear staining and rinsed with PBS for mounting and analysis. Microphotographs were taken using a confocal microscope.

De Filippis L., Halikere A., McGowan H., Moore J.C., Tischfield J.A., Hart R.P, & Pang Z.P. (2016). Ethanol-mediated activation of the NLRP3 inflammasome in iPS cells and iPS cells-derived neural progenitor cells. Molecular Brain, 9, 51.

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Protein Extraction and Western Blot Analysis

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Proteins were extracted from colon tissues using RIPA lysis buffer supplemented with proteinase and phosphatase inhibitors (Roche) as previously described.³⁰. Protein concentration was assessed using Pierce BCA protein assay (ThermoFischer Scientific) and all samples were normalized to 2μg/ml. Samples were resolved by 8%–15% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto polyvinylidene difluoride membranes. Blocking was performed in 5% skim milk for 1 h, and membranes were incubated with primary antibodies overnight at 4°C. Membranes were incubated with horse radish peroxidase–conjugated secondary antibody for 1 h, and proteins were visualized by using ECL substrate (ThermoScientific). Primary antibodies used were anti-pyrin (1:1000, ab195975, Abcam), anti-caspase-1 (1:500, sc-515, Santa Cruz Biotechnology), anti-P-STAT3 Tyr705 (1:1,000, 9131, Cell Signaling Technology), anti-IL-1β (1:1000, AF410, R&D systems) and anti-GAPDH (1:10,000; 5174, Cell Signaling Technology).

Sharma D., Malik A., Guy C.S., Karki R., Vogel P, & Kanneganti T.D. (2017). Pyrin Inflammasome Regulates Tight Junction Integrity to Restrict Colitis and Tumorigenesis. Gastroenterology, 154(4), 948-964.e8.

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Measuring Caspase-Induced Cytotoxicity in HEK-293T Cells

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HEK-293T cells were plated at 4 × 10⁵ cells in six-well plates and transfected using Fugene6 according to manufacturer's instructions (Promega, Madison, WI). Plasmids used included human TUBB6-Flag and TUBB-Flag (ThermoScientific), human caspase-1-Flag (variant alpha; OriGene, Rockville, MD), and human caspase-9 (variant alpha; OriGene). For assessing inhibition of caspase-induced cytotoxicity, 1 μg of total plasmid was used for each transfection, with 400 ng of caspase plasmid and 300 ng of TUBB6-Flag plasmid and the remainder made up with empty vector. Twenty-three hours posttransfection, cytotoxicity was measured using Cytotox-Glo. Cytotoxicity measurements were background subtracted (cells transfected with empty vector) and expressed as a percentage relative to caspase-1 or caspase-9 plus empty vector set to 100 to obtain “normalized RLUs.” Expression levels were assessed by Western blot for human caspase-1 (1:200, SC-515; Santa Cruz Biotechnology, Dallas, TX), caspase-9 (1:1000, C9; Cell Signaling Technology, Danvers, MA), and Flag (1:1000, M2; Sigma).

Salinas R.E., Ogohara C., Thomas M.I., Shukla K.P., Miller S.I, & Ko D.C. (2014). A cellular genome-wide association study reveals human variation in microtubule stability and a role in inflammatory cell death. Molecular Biology of the Cell, 25(1), 76-86.

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Immunoblotting for Active Caspase-1 Detection

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Immunoblotting was performed as described previously^{53 (link)}. THP-1 cell lines in suspension were treated with 50 ng/mL PMA (phorbol 12-myristate 13-acetate) for 12 h at 37 °C. For recovery of active caspase-1, cell culture supernatants of adherent THP-1 cells that were treated with one of the four compounds of interest were incubated with biotinyl-VAD-fmk (30 µM) overnight at 4 °C in order to precipitate the active, cleaved fragment p10 of caspase-1. The biotinyl-VAD-fmk/caspase-1 p10 complex was recovered by using streptavidin-Sepharose beads (Sigma), adding 30 µL of 1:1 streptavidin-Sepharose suspension per 250 mL of cell supernatant overnight at 4 °C. Beads were pelleted by centrifugation (3000 rpm/10 min/4 °C) and were washed 3 times in cold IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 50 mM NaF, 0.3% NP-40, 0.1 mM Na₃VO₄) before adding SDS loading buffer on top of the beads. Beads were then heated for 5 min and pelleted. Supernatant was analyzed by SDS-PAGE/immunoblotting using an antibody that detects the p10 small subunit of processed human caspase-1 (Santa Cruz Biotechnology, sc515).

Furman D., Chang J., Lartigue L., Bolen C.R., Haddad F., Gaudilliere B., Ganio E.A., Fragiadakis G.K., Spitzer M.H., Douchet I., Daburon S., Moreau J.F., Nolan G.P., Blanco P., Déchanet-Merville J., Dekker C.L., Jojic V., Kuo C.J., Davis M.M, & Faustin B. (2017). Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states. Nature medicine, 23(2), 174-184.

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Western Blot Analysis of Caspase-1 and IL-1β

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Western blot analyses were performed as previously described [10] . Caspase-1 [both the precursor peptide = procasp-1 (45 kDa) and the p10 subunit] and IL-1β [both the precursor peptide = proIL-1β (37 kDa) and the mature IL-1β (17 kDa)] were detected with rabbit polyclonal antibodies (sc-515 and sc-7884, respectively; Santa Cruz Biotechnology, Dallas, TX, USA). To confirm the homogeneity of the proteins loaded, the membranes were stripped, and incubated with an anti-βactin monoclonal antibody (Sigma-Aldrich). The membranes were overlaid with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Science, Norwalk, CT, USA), and exposed to Hyperfilm ECL film (Amersham Biosciences, Piscataway, NJ, USA).

Miglio G., Veglia E, & Fantozzi R. (2015). Fumaric acid esters prevent the NLRP3 inflammasome-mediated and ATP-triggered pyroptosis of differentiated THP-1 cells. International immunopharmacology, 28(1).

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