Ab8580
Manufactured by Merck Group
Ab8580 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, but its core function is not specified in the provided information. A more detailed and unbiased description cannot be provided while maintaining the requested criteria.
Automatically generated - may contain errors
Lab products found in correlation
Ab8895, by Abcam (2 mentions)
Ab4441, by Abcam (2 mentions)
Ab9050, by Diagenode (2 mentions)
H3k4me1, by Diagenode (2 mentions)
Protein a magnetic beads, by Thermo Fisher Scientific (2 mentions)
Cs 037 100, by Abcam (2 mentions)
H3k36me3, by Abcam (2 mentions)
H3k4me3, by Abcam (2 mentions)
Bioruptor, by Diagenode (2 mentions)
H3k27ac, by Abcam (2 mentions)
H3k9ac, by Abcam (2 mentions)
Ab4729, by Abcam (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Ab2237, by Abcam (1 mentions)
Ab817, by Abcam (1 mentions)
Chip it express kit, by Active Motif (1 mentions)
Hiseq sequencing system, by Illumina (1 mentions)
Ab8895, by Merck Group (1 mentions)
Ab4729, by Merck Group (1 mentions)
Ab129087, by Abcam (1 mentions)
13 protocols using ab8580
1
Chromatin Immunoprecipitation Protocol
2
Immunofluorescent Analysis of Histone Modifications
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Oocytes and embryos were fixed with 4% (w/v) paraformaldehyde for 40 min at room temperature, followed by three washes with DPBS containing 0.05% (w/v) polyvinyl alcohol and 0.01% (v/v) Tween-20. Samples were then permeabilized with 0.5% (v/v) Triton X-100 in DPBS for 60 min, blocked with 3% (w/v) BSA in DPBS for 2 h at room temperature, and incubated with primary antibodies for H3K4me3 (Abcam ab8580; 1:500 dilution) or H3K27me3 (Millipore 07-449; 1:500 dilution) overnight at 4°C. After three washes, samples were incubated with Dylight 549 goat anti-rabbit IgG (Abbkine A23320; 1:500 dilution) for 1 h at room temperature. After another three washes, samples were mounted on glass slides with anti-fade medium containing 4,6-diamidino-2-phenylindole (DAPI) and examined with a laser scanning confocal microscope (Zeiss LSM 510). The average fluorescence intensity per unit area within the region of interest was determined by ImageJ software (v1.48). Images were converted into eight-bit format to obtain the mean gray value of region of nuclei in each sample by auto threshold adjustment with default setting.
3
Chromatin Immunoprecipitation of Drosophila Larvae
4
Chromatin Immunoprecipitation Assay in mESCs and EBs
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Chromatin immunoprecipitation (ChIP) assays of mESCs and EBs were performed using a ChIP-IT Express Kit (Active Motif, 53008) following the manufacturer’s instructions. Briefly, the cells were cross-linked with 1% formaldehyde at room temperature for 10 minutes and quenched with 0.25 m glycine for 5 minutes. The cell pellet was incubated in lysis buffer on ice for 30 minutes and stroked to release nuclei. Chromatin was sheared to 150-500 bp fragments with a sonicator (Qsonica Q700) in Shearing Buffer. Immunoprecipitation was performed with Protein G Magnetic Beads and primary antibodies (anti-H3K4me3, Abcam#ab8580; anti-H3K27me3, Millipore#07-449; anti-H3K27Ac, Abcam#ab4729) by rotation at 4 °C overnight. Beads were washed thrice with ChIP Buffer 1 and twice with ChIP Buffer 2. Chromatin was eluted by rotation at room temperature for 15 minutes and reversed cross-linked at 65 °C for 4 h with Reverse Cross-linking Buffer. Then, the samples were incubated with RNase A at 37 °C for 15 minutes and Proteinase K at 42 °C for 1.5 h. DNA was purified using NucleoSpin Extract II Kit (MN, 740609).
5
Genomic Profiling with DNase-seq, ChIP-seq, and RNA-seq
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The DNase-seq experiments were performed as previously described [99 (link)], except that in the nuclei isolation steps, 2% PVP 40 (Solarbio, catalog number 822B047) was added to the nuclei isolation buffer and nuclei washing buffer to extract high-quality nuclei. DNase-seq libraries were constructed with two biological replicates for each sample and sequenced on the Illumina platform to generate 50-bp single-ended reads [99 (link)].
Chromatin immunoprecipitation (ChIP) was performed using published protocols with minor modifications [100 (link)]. As above, 2% PVP40 was added to the nuclei isolation buffer and nuclei washing buffer. Two commercial antibodies directed against H3K4me3 (abcam ab8580) and H3K27me3 (Millipore 07–449) were used to obtain immunoprecipitated DNA. ChIP-seq libraries were constructed as previously described, and sequenced using the paired-end 150 base pairs (PE150) strategy [13 (link), 100 (link)].
Total RNA for each sample was extracted using TRIzol reagent and sequenced on an Illumina HiSeq sequencing system. Six biological replicates were developed for RNA-seq for each sample.
Chromatin immunoprecipitation (ChIP) was performed using published protocols with minor modifications [100 (link)]. As above, 2% PVP40 was added to the nuclei isolation buffer and nuclei washing buffer. Two commercial antibodies directed against H3K4me3 (abcam ab8580) and H3K27me3 (Millipore 07–449) were used to obtain immunoprecipitated DNA. ChIP-seq libraries were constructed as previously described, and sequenced using the paired-end 150 base pairs (PE150) strategy [13 (link), 100 (link)].
Total RNA for each sample was extracted using TRIzol reagent and sequenced on an Illumina HiSeq sequencing system. Six biological replicates were developed for RNA-seq for each sample.
6
Histone ChIP-seq Analysis in Embryonic Tissue
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ChIP-seq data sets for four histone marks (H3K4me1, H3K4me3, H3K27ac, and H3K27me3—Abcam ab8895, ab8580, ab4729, and Millipore 07-449, respectively) were generated for E11.5 limbs, while for E11.5 forebrain we produced ChIP-seq for H3K4me1, H3K4me3, and H3K27ac and used a previously generated H3K27ac data set (Nord et al. 2013 (link)). Histone ChIP-seq was carried out using previously described protocols (Bernstein et al. 2006 (link)). DNA libraries were prepared, sequenced, and analyzed as described for FLAG ChIP-seq. All histone data sets were generated with an input control.
7
Histone Modification Analysis by Western Blot
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Whole-cell lysate was prepared in 3D-RIPA buffer. Protein (20 µg) was separated by 12% SDS–PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were blocked using 5% nonfat dry milk in PBS containing 0.05% Tween 20, and then incubated with primary antibody overnight at 4 °C. HRP-conjugated secondary antibody was used and detected using the Pierce ECL Western Blot Substrate. Antibodies were obtained from the following sources: H3K9Ac (Abcam, ab32129, 1:1000), H3K4Me2 (Abcam, ab32356, 1:5000), H3K4Me1 (Abcam, ab8895, 1:5000), H3K4Me3 (Abcam, ab8580, 1:5000), H3K14Ac (Millipore, 07–353, 1:5000), H3K18Ac (Millipore, 07–354, 1:5000), LSD1 (Abcam, ab17721, 1:500), HDAC1 (Abcam, ab19845, 1:1000), total H3 (Cell Signaling Technology, 4499 S, 1:1000 or Abcam, ab1791, 1:10,000), β-actin (Santa Cruz Biotechnology, sc47778, 1:2000), CoREST1 (BD Transduction Laboratories, 612146, 1:500), SIN3A (Abcam, ab129087, 1:1000), HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1:5000 for β-actin blots, 1:2000 for all others). Blots shown are representative of at least two independent experiments. Uncropped versions of Western blots are provided in the Supporting Information (Supplementary Figs. 32 and 33 ).
8
Chromatin Immunoprecipitation of Drosophila Larvae
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Third instar larva WID or EID isolated from Canton S flies were fixed, pooled in 700 μL and processed as described17 (link). Around 300 imaginal discs were used in these experiments. Trypsin treated cells from GFP transgenic flies were fixed after sorting for 10 minutes at room temperature and sonicated in a Diagenode Bioruptor for 15 minutes at high power in lysis buffer (1% SDS, 10 mM Tris HCl ph 8.0 and 2mM EDTA). Immunoprecipitations were performed in RIPA buffer. For L3 ChIPs and Imaginal Discs ChIPSeq experiments we used 1 μg of the corresponding antibody. For ChIPs in sorted cells we used 0.45 μg of anti-H3K4me3, 0.3 μg of anti-H3K36me3, 0.33 μg of anti-H3K27ac and 1 μg of anti-H3K27me3. For L3 time-specific ChIPs, 5 Canton S wall-wandering third instar larvae were disrupted, fixed and sonicated as indicated above. . Immunocomplexes were recovered with Invitrogen ProteinA magnetic beads for 2h. The beads were washed three times in RIPA or IP buffer, once in LiCl buffer and twice in TE17 (link). Primers used for Real-Time PCR are listed in Supplementary Table 11 . The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).
9
Western Blot Analysis of Stem Cell Markers
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Proteins were extracted using 1 × SDS lysis buffer (50 mM Tris-HCl [pH 6.8], 100 mM DTT, 2% SDS, and 10% glycerol) and separated by SDS-polyacrylamide gel electrophoresis. Then the proteins were transferred to a nitrocellulose (NC) membrane (Millipore) and blocked with 5% skim milk in TBST at room temperature for 1 h. The membranes were incubated with primary antibody overnight at 4 °C and horseradish peroxidase (HRP)-linked secondary antibody (CST, 7074) at room temperature for 1 h. The signals were detected by reacting with chemiluminescent HRP substrate (Millipore) and visualized using a chemiluminescent detector (FUJIFILM). The primary antibodies included the following: anti-FAM122A (customized by Abclonal), anti–PIP5K1B (Abnova, H00008395-A01), HRP–conjugated anti-α-tubulin (Proteintech, HRP-66031), HRP-conjugated anti-GAPDH (Proteintech, HRP-60004), anti-octamer binding transcription factor 4 (OCT4; Abcam, ab181557), anti-SOX2 (Abcam, ab92494; Abclonal, A0561), anti-Homeobox protein NANOG (NANOG; Abcam, ab214549), anti–H3K4me3 (Abcam, ab8580), anti-H3K27me3 (Millipore, 07-449), anti-H3K27ac (Abcam, ab4729), anti-histone H3 (Abcam, ab1791), anti-p-YAP (CST, 13008), and anti-YAP (CST, 14074).
10
Histone Modification Profiling in MLL-AF9 Leukemia
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