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Rbbp5

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Rbbp5 is a protein that plays a core role in the regulation of gene expression. It functions as a component of several chromatin-modifying complexes, contributing to the management of chromatin structure and accessibility.

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Lab products found in correlation

Rabbit polyclonal anti gapdh 2118, by Cell Signaling Technology (1 mentions) Peroxidase conjugated secondary antibodies for western blotting, by Merck Group (1 mentions) Dpy30, by Fortis Life Sciences (1 mentions) Ash2l, by Fortis Life Sciences (1 mentions) Anti gfp 6556, by Abcam (1 mentions) Mouse monoclonal anti lamp1 h4a3, by Santa Cruz Biotechnology (1 mentions) Fascin, by Santa Cruz Biotechnology (1 mentions) Set1a, by Fortis Life Sciences (1 mentions) Enhanced chemi luminescence (ecl), by Merck Group (1 mentions) Flag tag (flag), by Merck Group (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Bradford reagent, by Bio-Rad (1 mentions) Hsp90, by Santa Cruz Biotechnology (1 mentions) H3k27me3 07 449, by Merck Group (1 mentions) Bioruptor, by Diagenode (1 mentions)

5 protocols using rbbp5

1

Antibodies for Protein Analysis

Cited 2 times
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Rabbit polyclonal DPY30, RBBP5, ASH2L and BIG1 antibodies were from Bethyl Laboratories. Rabbit polyclonal anti-GFP (6556) and mouse monoclonal anti-M6PR (ab2733) were from Abcam. Mouse monoclonal anti-Snx1 was obtained from BD Transduction Laboratories. Mouse monoclonal anti-LC3 was obtained from MBL (clone 4E12, catalogue number M152-3). Rabbit polyclonal anti-spastin 86-340 and CD8 antibodies were produced as previously described (Seaman, 2004 (link); Connell et al., 2009 (link)). Mouse monoclonal anti-LAMP1 (H4A3) was obtained from Santa Cruz Biotechnology. Rabbit polyclonal anti-GAPDH (2118) was from Cell Signalling Technology. Alexa Fluor® 488 and 568 labelled secondary antibodies for immunofluorescence were obtained from Molecular Probes. Peroxidase conjugated secondary antibodies for western blotting were obtained from Sigma.
Newton T., Allison R., Edgar J.R., Lumb J.H., Rodger C.E., Manna P.T., Rizo T., Kohl Z., Nygren A.O., Arning L., Schüle R., Depienne C., Goldberg L., Frahm C., Stevanin G., Durr A., Schöls L., Winner B., Beetz C, & Reid E. (2018). Mechanistic basis of an epistatic interaction reducing age at onset in hereditary spastic paraplegia. Brain, 141(5), 1286-1299.
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2

Western Blot Analysis of Protein Expression

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Equal amounts of protein or equal volumes of elution fraction (FPLC) were subjected to electrophoresis through 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked overnight at 4 °C in PBS containing 5% dried milk then probed overnight at 4° with the indicated primary antibodies (Fascin, Santa cruz, sc-21743, 1:1000; pFascin, ECM biosciences, FP-2661, 1:1000; Flag, Sigma-Aldrich, F1804; Pol2, Santa-cruz, sc-899, 1:1000; RbBP5, Bethyl Laboratories, A300-109A, 1:1000; Set1A, Bethyl Laboratories, A300-289A, 1:1000; mTOR, cell signalling technologies, 2983, 1:1000; S6, cell signalling technologies, 2217, 1:1000; pS6, cell signalling technologies, 2215, 1:1000). Membranes were then washed three times with TBST and incubated for 1 h with secondary antibody coupled to HRP (Millipore, 1:10000) followed by 3 washes in TBST and then chemiluminescent detection (ECL, Millipore).
Saad A., Bijian K., Qiu D., da Silva S.D., Marques M., Chang C.H., Nassour H., Ramotar D., Damaraju S., Mackey J., Bismar T., Witcher M, & Alaoui-Jamali M.A. (2016). Insights into a novel nuclear function for Fascin in the regulation of the amino-acid transporter SLC3A2. Scientific Reports, 6, 36699.
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3

Nuclear Protein Extraction and Immunoprecipitation

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Whole cell nuclear extracts were prepared by lysing cells for 30 min at 4°C in protein extraction buffer (300 mM NaCl, 100 mM Tris pH 8, 0.2 mM EDTA, 0.1% NP40, 10% glycerol) with protease inhibitors and recovery of the supernatant following centrifugation. For immunoprecipitation, 3-5 μg antibody was added to protein extract and rotated for 1-2 hours at 4°C followed by addition of 20μl protein G Dynabeads, incubation for an additional 1 hour and washing in extraction buffer without glycerol. For western blotting, protein concentration was estimated with Bradford reagent (Biorad) and equal amounts of extract were run on a 4% SDS-PAGE gel and transferred to nitrocellulose membranes. Antibodies used in this study were: UTX (Bethyl, A302-374A), Rbbp5 (Bethyl, A300-109A), RNA Pol II 8WG16 (BioLegend, 920101), Mll3/4 (this study), Mll4 (Diagenode, C15310100).
Dorighi K.M., Swigut T., Henriques T., Bhanu N.V., Scruggs B.S., Nady N., Still CD I.I., Garcia B.A., Adelman K, & Wysocka J. (2017). Mll3 and Mll4 facilitate enhancer RNA synthesis and transcription from promoters independently of H3K4 monomethylation. Molecular cell, 66(4), 568-576.e4.
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4

ChIP-seq Protocol for Histone Modifications

Cited 2 times
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The following antibodies were generated in-house: anti-Set1B (47 (link)), anti-Mll2, anti-Set1A, anti-H3, anti-H3K4me1, anti-H3K4me2, and anti-H3K4me3. Other antibodies used in this study were H3 (CST #1B1B2), H3K4me3 (CST #9727), Rpb1 (CST #D8L4Y), HSP90 (Santa Cruz #7947), and Rbbp5 (Bethyl Laboratories #A300-109A). Set1A ChIP-seq data in Fig. 2B were from the Sze et al. (38 (link)).
Sze C.C., Ozark P.A., Cao K., Ugarenko M., Das S., Wang L., Marshall S.A., Rendleman E.J., Ryan C.A., Zha D., Douillet D., Chen F.X, & Shilatifard A. (2020). Coordinated regulation of cellular identity–associated H3K4me3 breadth by the COMPASS family. Science Advances, 6(26), eaaz4764.
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5

ChIP-seq Analysis of Chromatin Regulators

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ChIP was performed as previously described [56 (link)]. For CFP1, HCF1, RbBP5, hSET1 (SETD1A), Menin and UTX, chromatin was first cross-linked with ethylene glycol bis(succinimidyl succinate) (EGS) [29 (link)] in PBS at a final concentration of 2 mM for 60 min at RT. Formaldehyde (CH2O) was then added at a final concentration of 1% for 15 min at RT and samples were sonicated over 20 min (10 × 30-second episodes) at 4 °C to cleave genomic DNA (Bioruptor, Diagenode). Input data sets were matched with ChIP-seq samples. Antibodies used are: H3K27me3 (07-449) (Millipore); HCF1 (A301-400A), RbBP5 (A300-109A), hSET1 (SETD1A, A300-289A), Menin (A300-105A), UTX (A302-374A) from Bethyl Labs and CFP1 (CGBP, ab56035) from Abcam. The non-commercial CFP1 antibody was kindly provided by Prof. Robert Roeder. Real-Time PCR was performed using primers and probes (5’FAM-3’TAMRA) for the murine and human α-globin locus described previously [57 (link), 58 (link)]. Each ChIP was performed as two independent experiments and quality was assessed by qPCR. Libraries and sequencing (ChIP-seq) were performed using the standard Illumina kits and protocols.
van de Lagemaat L.N., Flenley M., Lynch M.D., Garrick D., Tomlinson S.R., Kranc K.R, & Vernimmen D. (2018). CpG binding protein (CFP1) occupies open chromatin regions of active genes, including enhancers and non-CpG islands. Epigenetics & Chromatin, 11, 59.
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