A ChIP-seq experiment was performed to determine the protein binding on CHK1 promoter. After knocking down FLT3-ITD in MV4-11 cells, the cells were collected and prepared for cross-linked chromatin. A Bioruptor UCD-200 ultrasound system (Diagenode) was used to break the chromatin into fragments of about 200 bp in size. The fragments were incubated with specific antibodies (anti-CBP (ab 119,488, Abcam), anti-p300 (ab14984, Abcam), anti-H3K9ac (ab272150, Abcam), anti-H3K27ac (ab203953, Abcam) at 4 degrees overnight. Rabbit serum was used as a control. High-Sensitivity ChIP Kit (ab185913, Abcam) was used for RNA elution and purification according to the manufacturer's instructions. QPCR was used to detect the degree of enrichment of the CHK1 promoter sequence.
Ab14984
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab14984 is a lab equipment product offered by Abcam. It is a device designed for laboratory use, but no further details about its core function can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab4729, by Abcam (4 mentions)
Ab7028, by Abcam (3 mentions)
Ab2832, by Abcam (3 mentions)
Ab203953, by Abcam (2 mentions)
High sensitivity chip kit, by Abcam (2 mentions)
Ab13537, by Abcam (2 mentions)
Ab13604, by Abcam (2 mentions)
Ab13888, by Abcam (2 mentions)
Bioruptor ucd 200 sonicator, by Diagenode (2 mentions)
Ab8895, by Abcam (2 mentions)
Ab 119 488, by Abcam (1 mentions)
Magna riptm rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Normal mouse igg, by Abcam (1 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Ab191250, by Abcam (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Ab170933, by Abcam (1 mentions)
Chamq sybr color qpcr master mix low rox premixed, by Vazyme (1 mentions)
Lightcycler480 real time pcr system, by Vazyme (1 mentions)
Chip it high sensitivity kit, by Active Motif (1 mentions)
25 protocols using ab14984
1
Determining Protein Binding on CHK1 Promoter
2
RNA Immunoprecipitation and qRT-PCR Analysis
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Cells were harvested and lysed with polysome lysis buffer (10 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP-40, 1 mM DTT, 100 U/ml RRI, 20 μl/ml protein inhibitor calculator, 2 mM vanadyl ribonucleotide complex solution). Then incubated with 2 μg HDAC2, HDAC1 (Abcam, ab7028) or p300 (Abcam, ab14984) antibody overnight at 4 °C. Then washed the non-specifically binding antibodies and incubated with Dynabeads protein A for 4 h at 4 °C. Then washed and precipitated the RNA with ethanol and sodium acetate. The RNA fraction was reverse transcription and analyzed with qRT-PCR.
3
RIP Assay for p300-MALAT1 Interaction
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RIP was applied to verify the interaction between p300 and MALAT1 using the Magna RIP TM RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). In brief, the pulmonary epithelial cells were lysed. A total of 100 μL cell extract was incubated with 900 μL RIP buffer containing magnetic bead coated by p300 rabbit antibody (ab14984, 1:50; Abcam, Cambridge, UK) or immunoglobulin G (IgG) for 3 h at 4°C. Beads were washed three times with lysis buffer, and RNA was extracted by addition of TRIzol to the beads. Finally, qPCR was performed to detect the MALAT1.
4
Profiling AML1 Genomic Occupancy
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Three biological replicate ChIP‐seq experiments were conducted for the specific detection of AML1‐bound genomic regions on THAP10 and miR‐383 promoters according to standard procedures with several modifications. Briefly, cross‐linked chromatin from approximately 5–10 × 107 SKNO‐1, SKNO‐1‐siA/E, U937 and U937‐A/E cells was prepared and fragmented to an average size of approximately 200 bp by 30–40 cycles of sonication (30 s each) in 15 ml tubes using the Bioruptor UCD‐200 sonicator (Diagenode). For immunoprecipitation, AML1 (sc‐8563, Santa Cruz Biotechnology), ETO (sc‐9737, Santa Cruz Biotechnology), HDAC1 (ab7028, Abcam), DNMT1 (ab13537, Abcam), DNMT3a (ab13888, Abcam), DNMT3b (ab13604, Abcam) and p300 (ab14984, Abcam) antibodies were added to 12 ml of diluted, fragmented chromatin. Nonimmunized rabbit serum served as a control. ChIP using the normal mouse IgG (Abcam) antibody was performed on naked and sonicated DNA extracted from the same cell samples and assayed using the EZ‐ChIP™ Chromatin Immunoprecipitation kit (Millipore) as per the manufacturer's instructions. Genomic THAP10 and pre‐miR‐383 upstream regions close to the putative AML1‐binding site were amplified. Primer sequences are shown in Appendix Table S7 . GAPDH served as a control for nonspecific precipitated sequences.
5
Chromatin Immunoprecipitation with Enzymatic Digestion
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Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP®
Enzymatic Chromatin IP Kit (#9003, CST, U.S.A.) according to the
manufacturer’s instruction. Cells were harvest at 80–90%
confluence after siRNA transfection. Briefly, cells were cross-linked with
1% formaldehyde for 15 min, and then terminated with glycine before
scrapped from culture dishes. Afterwards, the collected cells were digested with
nuclease to break the cross-linked chromatin to 100–200 bp length. Then
appropriate amount of chromatin was immunoprecipitated using anti-CBP (ab2832,
Abcam, U.S.A.), anti-P300 (ab14984, Abcam, U.S.A), anti-histone H3A (acetyl K27)
(ab4729, Abcam, U.S.A.), and anti-histone H3A (acetyl K18) (Cat.#17-10111,
Millipore, U.S.A.); goat anti-Rabbit IgG and goat anti-Mouse IgG were used as
negative control respectively. The immunoprecipitation reaction was performed
overnight at 4°C with rotation. The next day, the precipitated chromatin
were washed and eluted from the antibody/protein G magnetic beads, and then DNA
purification was performed and analyzed by qPCR with specific primers listed in
Table 1 .
Enzymatic Chromatin IP Kit (#9003, CST, U.S.A.) according to the
manufacturer’s instruction. Cells were harvest at 80–90%
confluence after siRNA transfection. Briefly, cells were cross-linked with
1% formaldehyde for 15 min, and then terminated with glycine before
scrapped from culture dishes. Afterwards, the collected cells were digested with
nuclease to break the cross-linked chromatin to 100–200 bp length. Then
appropriate amount of chromatin was immunoprecipitated using anti-CBP (ab2832,
Abcam, U.S.A.), anti-P300 (ab14984, Abcam, U.S.A), anti-histone H3A (acetyl K27)
(ab4729, Abcam, U.S.A.), and anti-histone H3A (acetyl K18) (Cat.#17-10111,
Millipore, U.S.A.); goat anti-Rabbit IgG and goat anti-Mouse IgG were used as
negative control respectively. The immunoprecipitation reaction was performed
overnight at 4°C with rotation. The next day, the precipitated chromatin
were washed and eluted from the antibody/protein G magnetic beads, and then DNA
purification was performed and analyzed by qPCR with specific primers listed in
6
RNA Immunoprecipitation Methodology
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RNA immunocoprecipitation was performed as previously described [43 (link), 44 (link)]. Briefly, cells were harvested and lysed with polysome lysis buffer (5 mM MgCl2, 10 mM KCl, 10 mM HEPES pH 7.0, 1 mM DTT, 0.5% NP-40, 100 U/ml RNase inhibitor, 20 μl/ml protein inhibitor calculator). Then lysed cells were incubated with 5 μg EZH2 (Abcam, ab191250) or P300 (Abcam, ab14984) antibody overnight at 4 °C. Next, using polysome lysis buffer to wash the non-specifically binding antibodies and incubated with Dynabeads protein G (Invitrogen, USA) for 4–6 h at 4 °C. Then washed and precipitated the RNA with ethanol and sodium acetate. The RNA fragments were reverse transcribed and analyzed by RT-qPCR.
7
ChIP-seq Analysis of PPM1G Regulation
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For the ChIP-seq reanalysis, the ChIP-seq datasets were obtained from the cistrome database (http://cistrome.org/db/ ). The binding of multiple transcription factor/co-regulators among the regulatory regions for PPM1G was plotted in the WashU browser.
The ChIP experiments were performed with the ChIP-IT high sensitivity kit (#53040, Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The antibodies against FOXA1 (#ab170933, Abcam), MYC (#9402, Cell Signaling Technology), MED1 (#17-10530, EMD Millipore, Temecula, CA, USA), and EP300 (#ab14984, Abcam) were used. The primers against the promoter regions and distal desert regions were listed as follows:
PPM1G promoter-F: 5′-GGGCAGTCATTTCCTTAGCA-3′; PPM1G promoter-R: 5′-GGGAACGAAGGAAGAGGTTC-3′; ChIP-negative-F:5′-CCCATCTTCTTTCCTGACCA-3′; ChIP-negative-R:5′-TCAGAGAAGCACCACCTCCT-3′. The qPCR was performed with the ChamQ SYBR Color qPCR Master Mix (Low ROX Premixed) (#Q431-02, Vazyme, China) in the Light Cycler® 480 Real-Time PCR System.
The ChIP experiments were performed with the ChIP-IT high sensitivity kit (#53040, Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The antibodies against FOXA1 (#ab170933, Abcam), MYC (#9402, Cell Signaling Technology), MED1 (#17-10530, EMD Millipore, Temecula, CA, USA), and EP300 (#ab14984, Abcam) were used. The primers against the promoter regions and distal desert regions were listed as follows:
PPM1G promoter-F: 5′-GGGCAGTCATTTCCTTAGCA-3′; PPM1G promoter-R: 5′-GGGAACGAAGGAAGAGGTTC-3′; ChIP-negative-F:5′-CCCATCTTCTTTCCTGACCA-3′; ChIP-negative-R:5′-TCAGAGAAGCACCACCTCCT-3′. The qPCR was performed with the ChamQ SYBR Color qPCR Master Mix (Low ROX Premixed) (#Q431-02, Vazyme, China) in the Light Cycler® 480 Real-Time PCR System.
8
Analyzing Transcriptional Regulators in Mycobacterial Infection
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Anti-LXRα (ab41902 Abcam), anti-ATF1 (sc270, Santa Cruz), anti-ATF3 (sc188, Santa Cruz), anti-ATF5 (sc377168, Santa Cruz), anti-RXRA (sc553, Santa Cruz), anti-RARA (sc551, Santa Cruz), anti-MEF2A (ab7606. Abcam), anti-MEF2C (ab64644, Abcam), anti-LC3 (NB100-2220, Novus), anti-β-actin (sc-1616, Santa Cruz), anti-H3K27ac (ab4729),anti-NCoR (ab24552), anti-KAT3B/p300 (ab14984), anti-KAT13A/SRC1/NCoA1 (ab84), anti-H3K4me1 (ab8895, abcam), anti-PPARγ (ab41928, Abcam), anti-Mtb-FITC (ab20962, Abcam), anti-rabbit Alexa647 (A-21245, Life Technologies), Prolong Gold with Dapi (Life Technologies), anti-rabbit-Alexa488 (Life Technologies), Bodipy 493 (Life Technologies). TO901317, ATRA and BAX Inhibiting Peptide V5 were bought from Sigma and resupended in DMSO. Nelfinavir (Nel) and Ritonavir (Rit) were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases. Auto Histone ChIP-seq Kit (Protein A) was bought from Diagenode.
9
Chromatin Immunoprecipitation Assay for Transcription Factors
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Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express kit (Active Motif)33 (link), according to the manufacturer’s instructions. In brief, HUVEC were cross-linked for 10 min with 1% formaldehyde. Chromatin was sheared using a Bioruptor UCD-200 ultrasound sonicator (Diagenode) and immunoprecipitated with 3 μg antibody to ERG (sc-354X, Santa Cruz Biotechnology), KLF2 (rabbit, ab203591, Abcam), H3K27Ac (rabbit, 39133, Active Motif) or p300 (mouse, ab14984, Abcam). The respective negative controls were rabbit IgG (PP64, Chemicon, Millipore) and mouse IgG (12- 371, Millipore). Immunoprecipitated DNA was then used as template for quantitative PCR using primers specific for genomic loci and listed in Supplementary Table 5 .
10
ChIP-PCR/qPCR Profiling of Transcriptional Regulators
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ChIP-PCR/qPCR was performed using the ChIP kit (ab500) from Abcam according to the manufacturer’s instructions. JEG3 cells were harvested, resuspended in PBS, and cross-linked using 1% formaldehyde. Glycine was added to stop the cross-linking reaction, and cells were washed twice with ice-cold PBS. Nuclei were isolated and then lysed using ice-cold Cell Lysis Buffer containing protease inhibitors and PMSF. Cross-linked chromatin was sheared using a Covaris S220 to 200- to 500-bp fragments and assessed by TapeStation assay D1000 (Agilent Technologies). Samples were then diluted with ChIP Dilution Buffer and incubated with antibodies anti-: Mock IgG1, IgG, ELF1, ELF5, RFX5 antibodies (Santa Cruz Biotechnology), ELF3 (A13489; Abclonal), BRD4 (A301-985A50) and MED1 (A300-793A) from Bethyl Laboratories, and MED23 (clone D27-1805; BD Biosciences), P300 (ab14984; Abcam), and H3K27Ac (ab4729, Abcam) overnight at 4 °C. The antibody/chromatin samples were pelleted and incubated with protein A beads. After incubation, the antibody/chromatin/beads were washed, and DNA was purified with DNA purifying slurry included in the ChIP kit. A total of 2 μL of DNA was used for qRT-PCR analysis using SYBR Green (Thermo Fisher Scientific) or 2 μl DNA/reaction was applied to PCR using platinum PCR SuperMix High Fidelity (Thermo Fisher Scientific) and analyzed by gel electrophoresis.
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