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Ab50558

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Ab50558 is a laboratory equipment product. It is a device used for scientific experiments and research purposes. The core function of this product is to perform a specific task related to laboratory activities.

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Lab products found in correlation

Ab156683, by Abcam (2 mentions) Ab12286, by Abcam (2 mentions) Ab5642, by Abcam (2 mentions) Ab14601, by Abcam (2 mentions) Nitrocellulose membrane, by GE Healthcare (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Ab50910, by Abcam (2 mentions) Xrn1 a300 443a, by Fortis Life Sciences (2 mentions) Antibody against zap 16820 1 ap, by Proteintech (2 mentions) Gfp g1546 antibody, by Merck Group (2 mentions) Dynabeads m 280 sheep anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Anti flag m2 magnetic bead, by Merck Group (1 mentions) Minelute kit, by Qiagen (1 mentions) Bioruptor, by Diagenode (1 mentions) Anti flag hrp, by Merck Group (1 mentions) Anti β actin, by Cell Signaling Technology (1 mentions) Ab63801, by Abcam (1 mentions) Ab26350, by Abcam (1 mentions) P0448, by Agilent Technologies (1 mentions) Donkey anti rabbit fitc, by Jackson ImmunoResearch (1 mentions)

6 protocols using ab50558

1

ChIP-qPCR Protocol for Exosome Complex

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The following antibodies were used: anti-EXOSC3 (ab156683; Abcam), anti-EXOSC10 (ab50558; Abcam), anti-DIS3 (PA5-34549; ThermoFisher Scientific), and anti-IgG (ab171870). ChIP experiments were conducted as described (Lee et al., 2006 (link)). For experiments with ChIP followed by qPCR, crosslinking was performed for 10 min. For sonication, we used a refrigerated Bioruptor (Diagenode), which we optimized to generate DNA fragments of approximately 200–1,000 base pair (bp). Lysates were pre-cleared for 2 hours using the appropriate isotype-matched control antibody (rabbit IgG; Abcam). The specific antibodies were coupled with magnetic beads (Dynabeads® M-280 Sheep Anti-rabbit IgG; ThermoFisher Scientific) overnight at 4°C. Antibody-bound beads and chromatin were then i mmunoprecipitated overnight at 4°C with rotation. For FLAG-ChIP, chromatin was immunoprecipitated using anti-FLAG M2 magnetic beads (Sigma). After washing, reverse crosslinking was carried out overnight at 65°C. After digestion with RNase and proteinase K (Roche), DNA was isolated with a MinElute kit (Qiagen) and used for downstream applications.
Rialdi A., Hultquist J., Morales D.J., Peralta Z., Campisi L., Fenouil R., Moshkina N., Wang Z.Z., Laffleur B., Michael R., Haas K., Pefanis E., Albrecht R.A., Pache L., Chanda S., Jen J., Ochando J., Byun M., Basu U., Garcia-Sastre A., Krogan N., van Bakel H, & Marazzi I. (2017). The RNA exosome syncs IAV-RNAPII transcription to promote viral ribogenesis and infectivity.. Cell, 169(4), 679-692.e14.
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2

Western Blot Analysis of Exosome Proteins

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Antibodies used are as follows: anti–β-actin (Cell Signaling, 3700); anti-EXOSC3 (ab156683; Abcam), anti-EXOSC10 (ab50558; Abcam), anti-DIS3 (PA5-34549; ThermoFisher Scientific), anti-RBM7 (HPA013993, Sigma) anti-HNRNPA2 (Cell Signaling, 9304), anti-FLAG-HRP (A8592; Sigma), anti-influenza A M1 (P. Palese laboratory), and anti-influenza A H1N1 HA (ThermoFisher Scientific, PA5-20713). Gradient gels (4-12%) were used according to the molecular weight of the proteins to be evaluated, followed by wet transfer on polyvinylidene fluoride membranes.
Rialdi A., Hultquist J., Morales D.J., Peralta Z., Campisi L., Fenouil R., Moshkina N., Wang Z.Z., Laffleur B., Michael R., Haas K., Pefanis E., Albrecht R.A., Pache L., Chanda S., Jen J., Ochando J., Byun M., Basu U., Garcia-Sastre A., Krogan N., van Bakel H, & Marazzi I. (2017). The RNA exosome syncs IAV-RNAPII transcription to promote viral ribogenesis and infectivity.. Cell, 169(4), 679-692.e14.
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3

Western Blot Protocol for Protein Detection

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Cells were counted, normalized for cell number, lysed in SDS sample buffer, separated by electrophoresis on NuPage 4–12% Bis-Tris gels (Novex) and blotted onto nitrocellulose membranes (GE Healthcare). Antibodies for PTBP1 (ab5642), Drosha (ab12286), DICR (ab14601), EXOSC6 (ab50910), EXOSC10 (ab50558), and PARN (ab188333) were obtained from Abcam. Antibodies for Upf1 (A300-036A), METTL3 (A301-567A), EXOSC4 (A303-775A), EXOSC5 (A303-887A), and Xrn1 (A300-443A) were obtained from Bethyl Labs. The antibody against ZAP (16820-1-AP) was obtained from Proteintech. The HIV-1 capsid antibody (183-H12-5C) was obtained from the NIH AIDS reagent repository. The GFP (G1546) antibody was obtained from Sigma. The HIV Env (12-6205-1) antibody was obtained from American Research Products. The HA (HA.11) antibody used in the CLIP assays was obtained from Biolegend.
Takata M.A., Gonçalves-Carneiro D., Zang T., Soll S.J., York A., Blanco-Melo D, & Bieniasz P.D. (2017). CG-dinucleotide suppression enables antiviral defense targeting non-self RNA. Nature, 550(7674), 124-127.
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4

Antibodies for Western Blot and Immunofluorescence

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The primary antibodies used for western blotting and immunofluorescence were: mouse anti-EXOSC10 (sc-374595, Santa Cruz Biotechnology, dilution 1:100 for IF and 1:1000 for WB), rabbit anti-EXOSC10 (ab50558, dilution 1:500), rabbit anti-DIS3 (ab176802, Abcam, dilution 1:100), mouse anti-Tubulin (Sigma, T5168, dilution 1:50), rabbit anti-γH2AX (#9718, Cell Signaling Technology, dilution 1:400), mouse anti-γH2AX (ab26350, Abcam, dilution 1:500), rabbit anti-RAD51 (ab63801, Abcam, dilution 1:100), mouse anti-RPA (MABE285, Merck Millipore, dilution 1:500), mouse anti-CtIP (61141, Active Motif, dilution 1:400), rat anti-BrdU (B8434, Sigma-Aldrich, dilution 1:250), and mouse monoclonal anti-BrdU (RPN20AB, Sigma, dilution 1:100). Fluorophore-conjugated secondary antibodies used for immunofluorescence were: goat anti-mouse Alexa594 (115-585-003, Jackson Immunoresearch, dilution 1:100), donkey anti-rabbit-FITC (711-096-152, Jackson Immunoresearch, dilution 1:100), goat anti-rat Alexa647 (112-605-143, Jackson Immunoresearch, dilution 1:100). Secondary antibodies for western blotting were: HRP-conjugated goat anti-mouse (P0447, Dako, dilution 1:1000) and HRP-conjugated goat anti-rabbit (P0448, Dako, dilution 1:2000).
Domingo-Prim J., Endara-Coll M., Bonath F., Jimeno S., Prados-Carvajal R., Friedländer M.R., Huertas P, & Visa N. (2019). EXOSC10 is required for RPA assembly and controlled DNA end resection at DNA double-strand breaks. Nature Communications, 10, 2135.
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5

Immunostaining of Meiotic Chromosome Spreads

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The surface spreads were prepared as described in a previous study using testicular samples from 12-week old adult C57BL/6jRj male mice60 (link). The spreads were co-immunostained for phosphorylated histone H2AX (γH2AX; green, 05-636, Millipore), and SYCP3, a protein in the axial element of the synaptonemal complex (red, sc-74569, Santa Cruz) or EXOSC10 (red, ab50558, Abcam). The images were taken using an AxioImager microscope equipped with an AxioCam MRc5 camera and AxioVision software version v4.8.1 (Zeiss, Le Pecq, France).
Jamin S.P., Petit F.G., Kervarrec C., Smagulova F., Illner D., Scherthan H, & Primig M. (2017). EXOSC10/Rrp6 is post-translationally regulated in male germ cells and controls the onset of spermatogenesis. Scientific Reports, 7, 15065.
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6

Western Blot Protocol for Protein Detection

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Cells were counted, normalized for cell number, lysed in SDS sample buffer, separated by electrophoresis on NuPage 4–12% Bis-Tris gels (Novex) and blotted onto nitrocellulose membranes (GE Healthcare). Antibodies for PTBP1 (ab5642), Drosha (ab12286), DICR (ab14601), EXOSC6 (ab50910), EXOSC10 (ab50558), and PARN (ab188333) were obtained from Abcam. Antibodies for Upf1 (A300-036A), METTL3 (A301-567A), EXOSC4 (A303-775A), EXOSC5 (A303-887A), and Xrn1 (A300-443A) were obtained from Bethyl Labs. The antibody against ZAP (16820-1-AP) was obtained from Proteintech. The HIV-1 capsid antibody (183-H12-5C) was obtained from the NIH AIDS reagent repository. The GFP (G1546) antibody was obtained from Sigma. The HIV Env (12-6205-1) antibody was obtained from American Research Products. The HA (HA.11) antibody used in the CLIP assays was obtained from Biolegend.
Takata M.A., Gonçalves-Carneiro D., Zang T., Soll S.J., York A., Blanco-Melo D, & Bieniasz P.D. (2017). CG-dinucleotide suppression enables antiviral defense targeting non-self RNA. Nature, 550(7674), 124-127.
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