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Ab156683

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Ab156683 is a laboratory product manufactured by Abcam. It is a piece of equipment designed for use in scientific research and laboratory settings.

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Lab products found in correlation

Ab50558, by Abcam (2 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (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) Silencer select pre designed sirna targeting, by Thermo Fisher Scientific (1 mentions) Bis tris gel, by Thermo Fisher Scientific (1 mentions) Immobilon p pvdf 0.45 mm membrane, by Merck Group (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)

3 protocols using ab156683

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

Exosomal RNA Knockdown Using siRNA

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For siRNA treatment, cells (GM10253A and HybNeo3; 10–20% confluent) were transfected with 10 nM Silencer Select Pre-designed siRNA targeting EXOSC3 (Ambion, Life Technologies) using Lipofectamine RNAi MAX (ThermoFisher) 24 h after seeding and again 48 h later. After a further 48 h exosome knockdown was confirmed by western blotting and TTseq was performed. Silencer Select RNA sequence for EXOSC3 were GAGATATATTCAAAGTTGA, part number s83102. The control RNA was Stealth RNAi siRNA Negative Control (ThermoFisher). For western blotting cells were suspended in NuPAGE LDS sample buffer (ThermoFisher) with 10 mM DTT, incubated at 100 °C for 5 min and sonicated briefly. Protein samples were resolved on 12% bis-tris gels (ThermoFisher) and transferred to Immobilon-P PVDF 0.45 mm membrane (Merck Millipore) by wet transfer. Membranes were probed with anti-EXOSC3 antibody (Abcam, Ab156683; 1:1000) using standard techniques and detected by enhanced chemiluminescence.
Naughton C., Huidobro C., Catacchio C.R., Buckle A., Grimes G.R., Nozawa R.S., Purgato S., Rocchi M, & Gilbert N. (2022). Human centromere repositioning activates transcription and opens chromatin fibre structure. Nature Communications, 13, 5609.
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