Protein extracts were separated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with antibodies against Daxx (M-112, Santa Cruz Biotechnology, 1:500), Atrx (H-300, Santa Cruz Biotechnology, 1:1,000), β-actin (A1978, Sigma, 1:5000) and H3.3 (Ab176840, Abcam, 1:1000). Proteins were visualized using Li-COR secondary antibodies and imaged using a ChemiDoc System (Bio-Rad). Protein levels were quantified in ImageJ and normalized to the level in the first lane.
Ab176840
Manufactured by Abcam
Sourced in United States
Ab176840 is a lyophilized antibody product. The core function of this product is to bind to a specific target.
Automatically generated - may contain errors
Lab products found in correlation
Ab1791, by Abcam (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
H 300, by Santa Cruz Biotechnology (1 mentions)
M 112, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Ab133327, by Abcam (1 mentions)
Sc 40, by Santa Cruz Biotechnology (1 mentions)
A4416, by Merck Group (1 mentions)
A6154, by Merck Group (1 mentions)
Complete mini edta, by Roche (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Ab6721, by Abcam (1 mentions)
Protein g agarose beads, by Cell Signaling Technology (1 mentions)
Proteinase k, by Takara Bio (1 mentions)
H3k27me3 9733s, by Cell Signaling Technology (1 mentions)
Ethachinmate, by Nippon Gene (1 mentions)
Ab6326, by Santa Cruz Biotechnology (1 mentions)
Ab9050, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
8 protocols using ab176840
1
Quantifying Chromatin Protein Levels
2
SDS-PAGE and Immunoblotting Protocol
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SDS–PAGE and immunoblotting was performed as described previously5 (link). In brief, cell pellets were lysed in RIPA buffer containing cOmplete Mini EDTA free (Roche) on ice for 20 min. Samples were then sonicated in a water-cooled Bioruptor Pico (Diagenode) and centrifuged at 21,000g for 15 min at 4 °C. Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Forty micrograms of protein was loaded per well. The primary antibodies were anti-CDK1 (1:1,000, ab133327, Abcam), anti-Myc (1:1,000, sc-40, Santa Cruz) and anti-histone H3.3 (1:5,000, ab176840, Abcam). The secondary antibodies were anti-mouse IgG peroxidase conjugate (1:10,000, A4416, Sigma-Aldrich) and anti-rabbit IgG peroxidase conjugate (1:10,000, A6154, Sigma-Aldrich).
3
Immunoblot Analysis of H3.3 and H3
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4
Chromatin Immunoprecipitation (ChIP) Protocol
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ChIP was also performed as described previously [4 (link)]. Chromatin samples extracted from each group were diluted to contained around 700 ng DNA. Chromatin was incubated overnight at 4°C with antibodies diluted at 1:50 for: anti-H3.3 (ab176840, Abcam, Cambridge, UK), anti-pan-acetyl H3 (39139, Active Motif, Carlsbad, CA), anti-H3 mono-methylated at lysine 4 (H3K4me1, 5326S, Cell Signaling Technology, Danvers, MA), anti-H3 tri-methylated at lysine 27 (H3K27me3, 9733S, Cell Signaling Technology), or anti-total H3 (4620S, Cell Signaling Technology), followed by subsequent reaction with protein G agarose beads (9007, Cell Signaling Technology; 20 μl for each reaction) for 4 h at 4°C. Beads were washed and incubated with proteinase K (Takara Bio, Shiga, Japan) for 1 h at 65°C. DNA was extracted by adding phenol-chloroform solution (25:24) and by centrifugation at 12,000 × g for 10 min at 20°C. The supernatant was collected, and ethanol precipitation was performed using Ethachinmate (NIPPON GENE). The final pellet was resuspended in tris-EDTA buffer and stored at −20°C. The amount of input DNA in the chromatin utilized for ChIP reaction was estimated with the same procedure but without any antibodies.
5
Chromatin Immunoprecipitation Protocol
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Antibodies used were directed against anti-H3 (Abcam, #ab1791), anti-H3.3 (Abcam, #ab176840), anti-H3K9me3 (Abcam, ab8898), anti-H3K36me3 (Abcam, #ab9050), anti-γH2A.X/phospho-histone H2A.X (Ser139) clone JBW301 (Merck Millipore, #05-636), anti-ATRX (Santa Cruz Biotechnologies, #sc15408), anti- KDM4B (Abcam, #ab191434) anti-IDH1 (Sigma Aldrich, SAB4100064), anti-IDH1R132H (Sigma Aldrich, SAB4200548), anti-HP1α (Merck Millipore, #MAB3584), anti-PML (Merck Millipore, #MAB3738), anti-TP53 (Cell Signaling Technologies, #cst-2524), anti-Flag (Sigma, #F1804), anti-TERF1 (Alpha Diagnostics, #TRF12-S) and anti-BrdU (Abcam, #ab6326), Anti-β Actin (AC-15) (Santa Cruz Biotechnology, #sc69879), Goat anti Rabbit IgG, HRP conjugate (Merck Millipore, #AP187P), Donkey anti-Mouse IgG HRP conjugate (Merck Millipore, #AP192P), Donkey anti-Mouse IgG (H + L) Alexa Fluor 594 (Invitrogen, #A-21203), Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen, #A-21206).
6
Chromatin Immunoprecipitation of Histone H3.3 Variants
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KNS-42 cells were fixed with 1% formaldehyde and incubated at room temperature for 15 min. The cells were collected with centrifugation and re-suspended in ChIP-lysis buffer (50 mM HEPES-KOH, pH7.5, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) sodium deoxycholate, 1% (v/v) NP-40, 0.1% (w/v) sodium dodecyl sulfate) containing 1 × protease inhibitor cocktail (Nacalai Tesque). The chromatin was sheared by sonication with Focused-ultrasonicator S220 (Covaris) using milliTUBE 1-ml AFA Fiber. For ChIP, anti-histone H3.3 rabbit monoclonal antibody (Abcam, ab176840) and anti-histone H3.3 G34V rabbit monoclonal antibody were used as primary antibodies with pre-washed Dynabeads Protein A (Dynal). The epitope of the former antibody lies between the 50th and C-terminal residues of human histone H3.3. The ChIP-ed DNA was purified using the DNeasy Blood and Tissue kit (Qiagen) with some modifications, and 10 ng of the DNA was used for library preparation with the SMARTer ThruPLEX DNA-seq Kit (Takara Bio) according to the manufacturer's instruction. Sequencing was performed using the HiSeq X Ten or NovaSeq 6000 at Macrogen Japan Corp. ChIP-seq reads were mapped to the reference human genome GRCh37 (hg19) and peak calling was performed using MACS242 (link).
7
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 2ΔCt method and shown as percentage relative to input DNA in the bar graphs.
8
Immunohistochemical Analysis of Histone Modifications
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Anti-H3.3antibody(#ab176840), anti-H3 antibody(#ab1971), anti-biotin antibody(#ab1227), anti-H3K4me3(#ab185637), and anti-H3K9me3(#ab8898) were purchased from Abcam (USA). GDNF was purchased from Peprotech (#450-44, USA). Male C57BL/6J mice were purchased from SIPPR-BK Animal Company (Shanghai, China).
The C18-4 cells was kindly provided by Prof. Zuping He, who was a principal investigator in Renji-Med X Clinical Stem Cell Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. The C18-4 cells were grown as described by He et al. [25 (link)].
The C18-4 cells was kindly provided by Prof. Zuping He, who was a principal investigator in Renji-Med X Clinical Stem Cell Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. The C18-4 cells were grown as described by He et al. [25 (link)].
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