Ab52484

Manufactured by Abcam

Ab52484 is a laboratory reagent. It is a protein that is used in various research applications.

Automatically generated - may contain errors

Lab products found in correlation

Ab1791, by Abcam (2 mentions) Ab10478, by Abcam (1 mentions) Ab26322, by Abcam (1 mentions) G6539, by Merck Group (1 mentions) Formaldehyde, by Electron Microscopy Sciences (1 mentions) T7451, by Abcam (1 mentions) T5192, by Santa Cruz Biotechnology (1 mentions) Anti acetyl histone h4, by Merck Group (1 mentions) Ab88770, by Abcam (1 mentions) α tubulin antibody t5168, by Merck Group (1 mentions) Ab47915, by Abcam (1 mentions) Ab176840, by Abcam (1 mentions) Ab8898, by Abcam (1 mentions) Dnase 1, by Promega (1 mentions) Polybrene, by Merck Group (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Protein a g beads, by Merck Group (1 mentions) Protein a g bead, by Santa Cruz Biotechnology (1 mentions)

5 protocols using ab52484

Immunofluorescence Labeling of Cellular Targets

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Mouse monoclonal antibodies used were anti-GFP ([1:100] Sigma-Aldrich, G6539), acetylated tubulin ([1:1,000] Sigma-Aldrich, T7451) and anti-histone H2B ([1:50] Abcam, ab52484) antibodies. Rabbit polyclonal antibodies used were anti-γ-tubulin ([1:1,000] Sigma-Aldrich, T5192), anti-lamin A/C ([1:20] Santa Cruz Biotechnology, H110), anti-cAMP protein kinase catalytic subunit ([1:1,000] Abcam, ab26322) and anti-CBX/HP1 beta antibodies ([1:100] Abcam, ab10478). TRITC or Alexa Fluor 647 conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc. or Life Technologies, respectively) were used for indirect immunofluorescence detection. BG-conjugated dyes, including SNAP-Surface Alexa Fluor 647 and 546 (NEB), were used for staining SNAP-tag expressed in cells.
The cells were fixed with 4% formaldehyde (Electron Microscopy Sciences) for 15 min, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS), stained for primary antibodies and Alexa-Fluor-conjugated secondary antibodies, or BG-conjugated fluorophores for SNAP-tag at 4 °C overnight.

Kamiyama D., Sekine S., Barsi-Rhyne B., Hu J., Chen B., Gilbert L.A., Ishikawa H., Leonetti M.D., Marshall W.F., Weissman J.S, & Huang B. (2016). Versatile protein tagging in cells with split fluorescent protein. Nature Communications, 7, 11046.

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CD4+ T Cell Histone Acetylation Analysis

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Ten million primary CD4⁺ T cells were treated with GTX concentration gradient, SAHA, or left untreated for 4 hours and then washed twice in PBS. Cells were lysed for 10 min at 4°C in TBE (Tris-borate-EDTA) buffer [PBS, 0.5% Triton X-100 (v/v), 2 mM phenylmethylsulfonyl fluoride, and 0.02% (w/v) NaN₃] at a density of 10⁷ cells per 1 ml of the buffer. Samples were centrifuged at 425g for 10 min at 4°C. Supernatants were discarded, and cell pellets were washed in half the volume of TEB buffer used for lysis and centrifuged as before. Supernatants were discarded, and pellets were resuspended in 0.2 N HCl at a density of 4 × 10⁷ cells/ml. Histones were extracted overnight at 4°C and then centrifuged at 425g for 10 min at 4°C. Supernatants were collected, protein concentration was determined by Bradford assay, and samples were subjected to SDS-PAGE Western blot. The following antibodies were used in Western blot analysis: anti–acetyl-histone H4 (06-598, Millipore) and anti-histone H2B (ab52484, Abcam).

Stoszko M., Al-Hatmi A.M., Skriba A., Roling M., Ne E., Crespo R., Mueller Y.M., Najafzadeh M.J., Kang J., Ptackova R., LeMasters E., Biswas P., Bertoldi A., Kan T.W., de Crignis E., Sulc M., Lebbink J.H., Rokx C., Verbon A., van Ijcken W., Katsikis P.D., Palstra R.J., Havlicek V., de Hoog S, & Mahmoudi T. (2020). Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency. Science Advances, 6(33), eaba6617.

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Histone Expression Analysis in C. elegans

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Worms were synchronized by bleaching and plated to RNAi plates as L1 larvae. Worms were collected from plates at L4 stage with M9 solution and freezed immediately in liquid nitrogen minimizing the time worms stayed in the solution. Worms were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0,1% sodium dodecyl sulfate (SDS)) with sonication. Lysates from L4 larvae worms were resolved by SDS–polyacrylamide gel electrophoresis. Histone H2A (ab88770, used with 1:400 dilution), H2B (ab52484, used with 1:1000 dilution), and H3 (ab1791, used with 1:1000 dilution) antibodies were purchased from Abcam, histone H4 antibody (sc-10810, used with 1:400 dilution) from Santa Cruz, and α-tubulin antibody (T5168, used with 1:1000 dilution) from Sigma. Uncropped membrane slices are shown in Supplementary Fig. 6.

Matilainen O., Sleiman M.S., Quiros P.M., Garcia S.M, & Auwerx J. (2017). The chromatin remodeling factor ISW-1 integrates organismal responses against nuclear and mitochondrial stress. Nature Communications, 8, 1818.

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Chromatin Immunoprecipitation for Histone Analysis

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Cells were seeded (1.5 to 2 × 10⁶ per 10-cm dish) one day before infection. Virus supernatants (15 µL) were pretreated with 5 U/mL DNase I (Promega, M6101) for 1 h at 37 °C. Medium was supplemented with 8 µg/mL Polybrene (Millipore-Sigma, TR-1003-G). Virus and Polybrene were removed after 5 h. Cells were washed twice and harvested for ChIP ∼24 h after infection. ChIP protocol was performed as previously described (11 (link)). Next, ChIP-grade antibodies were used with a concentration of 5 µg per 50 µg sonicated chromatin: anti-H1.2 ([EPR12690]; #ab181973; Abcam); anti-H1.4 ([D4J5Q]; #41328S; Cell Signaling Technology); anti-H2B (#ab52484; Abcam); anti-H3 (#ab1791; Abcam); anti-H3.3 ([EPR17899]; #ab176840; Abcam); anti-H3K9me3 (#ab8898; Abcam); anti-H3 acetyl K9+K14+K18+K23+K27 (#ab47915; Abcam); rabbit IgG isotype control (#02–6102; Invitrogen); and mouse IgG1 isotype control ([G3A1]; #5415S; Cell Signaling Technology). After DNA purification, 5 µL per sample were used for qPCR analysis. PCR protocol and primers were used as described above in “Viral DNA Detection.” β-Globin–specific primers served as a heterochromatin control (for 5′-CAGAGCCATCTATTGCTTAC-3′, rev: 5′-GCCTCACCACCAACTTCATC-3′). Data were calculated relative to the respective input DNA with the 2^ΔCt method and shown as percentage relative to input DNA in the bar graphs.

Geis F.K., Sabo Y., Chen X., Li Y., Lu C, & Goff S.P. (2022). CHAF1A/B mediate silencing of unintegrated HIV-1 DNAs early in infection. Proceedings of the National Academy of Sciences of the United States of America, 119(4), e2116735119.

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IL-33 Chromatin Binding Assay

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Necrosis was induced by cryoshock in pools of TE-7 cells with Dox-inducible overexpression of WT IL-33 as described above in PBS. Protein concentration was determined by BCA assay (23227, Thermo Scientific). Equal amounts of proteins were precleared for 2 h at 4 °C with Protein A/G beads (sc-2003, Santa Cruz) in the presence of 250 mM NaCl, 1% NP-40, 1 mM EDTA, and protease inhibitors. Samples were incubated overnight at 4 °C with 2 µg of antibody. Samples were incubated for 1 h at 4 °C with Protein A/G beads before elution with glycine (pH 2.8). Protein expression in eluates was determined by western blot analysis.
In separate experiments, 4 µg each of recombinant GST-tagged full-length IL-33 (H00090865-P01, Abnova) and acid-purified histones were incubated together in 1 mL of PBS, treated with DNase, and incubated for 1 h with mouse anti-histone H2B (ab52484, Abcam) and rabbit anti-histone H2A (07–146, Millipore) or control IgG for 1 h, and then incubated overnight at 4 °C in Protein A/G beads precleared with PBS. Elution was performed with 1 M glycine (pH 2), and protein expression in eluates was determined by western blot analysis.

Travers J., Rochman M., Miracle C.E., Habel J.E., Brusilovsky M., Caldwell J.M., Rymer J.K, & Rothenberg M.E. (2018). Chromatin regulates IL-33 release and extracellular cytokine activity. Nature Communications, 9, 3244.

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