The preparation of ChIP and input DNA libraries was performed as previously described [32 (link)]. In brief, two cells were crosslinked with 1% formaldehyde for 5 min at room temperature and quenched with glycine (125 mM). Cells were then put on ice, resuspended in cold cell lysis buffer [140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS, and protease inhibitors (Roche)]. Nuclei were sonicated into fragments of 200–1000 bp in size. The chromatin fragments were precleared and then immunoprecipitated with Protein A + G magnetic beads coupled with anti-H3K4me3 (ab8580; Abcam), anti-H3K27ac (ab4729; Abcam), anti-H3K27me3 (07-449; Millipore), and anti-CTCF (ab70303; Abcam). After reverse crosslinking, immunoprecipitated DNA and input DNA were end-repaired and adapters were ligated to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module (E7442; NEB) and NEBNext Ultra Ligation Module (E7445; NEB). High-throughput sequencing of the ChIP fragments was performed using Illumina NextSeq 500, following the manufacturer’s protocol.
Ab70303
Manufactured by Abcam
Sourced in United States
Ab70303 is a mouse monoclonal antibody that recognizes the CD3 epsilon chain. CD3 is a protein complex associated with the T cell receptor and is involved in T cell activation.
Automatically generated - may contain errors
Lab products found in correlation
Ecl system, by Cytiva (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Sds page, by Thermo Fisher Scientific (3 mentions)
Ab992, by Abcam (3 mentions)
Am4300, by Proteintech (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab9263, by Abcam (2 mentions)
Ab5408, by Abcam (2 mentions)
Nextseq 500, by Illumina (1 mentions)
Ab4729, by Abcam (1 mentions)
Ab8580, by Abcam (1 mentions)
Nebnext ultra end repair da tailing module, by New England Biolabs (1 mentions)
Nebnext ultra ligation module, by New England Biolabs (1 mentions)
Ab31387, by Abcam (1 mentions)
Ab8895, by Abcam (1 mentions)
Ab26721, by Abcam (1 mentions)
Ab9050, by Abcam (1 mentions)
Sc 352x, by Santa Cruz Biotechnology (1 mentions)
Ab1791, by Abcam (1 mentions)
α tubulin t6199, by Merck Group (1 mentions)
15 protocols using ab70303
1
ChIP-seq Library Preparation Protocol
2
Protein Expression Analysis via Western Blot
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Cell lysates were prepared in RIPA buffer, subjected to SDS-PAGE (Thermo Fisher Scientific), and transferred to PVDF membranes (Bio-Rad) at 100 V for 1 h. After blocking, the membranes were incubated with 5% nonfat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) containing antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), USF1 (Proteintech, 22327–1-AP, 1:2000), USF2 (Novus, NBP1-92649, 1:2000), and CTCF (Abcam, ab70303, 1:1000) at 4 °C for 12 h with gentle shaking. The membranes were washed three times for 30 min and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 2 h at room temperature. The blots were washed with TBS-T three times for 30 min and developed with the ECL system (Amersham Biosciences).
3
ChIP-qPCR for Histone Modifications
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ChIP was performed as described before.26 (link) In brief, cells were fixed in 0.8% formaldehyde for 6 min at room temperature. Sonicated chromatin was immunoprecipitated with antibodies for H3K4me1 (ab8895; Abcam, Cambridge, MA, USA), H3K36me3 (ab9050; Abcam), RNA polymerase II CTD (ab26721; Abcam), Rad21 (ab992; Abcam), PU.1 (sc-352X; Santa Cruz), CREB (ab31387; Abcam), Hoxa9 (sc-17155X; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CTCF (ab70303; Abcam), or rabbit IgG (15006; Sigma-Aldrich). A 10% aliquot was removed as an input fraction. Quantitative real-time PCR was performed with ChIP DNA and input DNA using a Light Cycler 480II. Relative quantitation was carried out by the comparative threshold cycle (CT) method. Statistical analysis was performed using the GraphPad Prism 5 software. The Student's t-test was used on measurements of enrichment from different condition samples from three experimental replicates. Primers for ChIP-qPCR are shown in Table 1 .
4
Western Blot Analysis of Proteins
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Cells lysate was prepared by using RIPA buffer followed with SDS-PAGE (Thermo Fisher Scientific) and transferred to a PVDF membrane according to the manufacturer’s protocols (Bio-Rad) at constant 100 V for 1 h. After blocking incubation with 5% non-fat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 1 h at room temperature, the membrane was incubated with antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), AID (MBL, M3-214-3, 1:2,000), MYC (Cell Signaling Technology, #9402, 1:1,000), ACTIN (Sigma, MABT825, 1:5,000) and CTCF (abcam, ab70303, 1:1,000) at 4°C for 12 h with gentle shaking. Membranes were washed three times for 30 min and incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 2 h at room temperature. Blots were washed with TBS-T three times for 30 min and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocol.
5
Comprehensive Antibody Panel for Chromatin Studies
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Antibodies recognizing SMC2 (A300-058A), SMC4 (A300-064A), NCAPH2 (A302-275A), NCAPD3 (A300-604A), NCAPG2 (A300-605A), TFIIIC-220 (A301-291A), TFIIIC-102 (A301-238A), TFIIIC-63 (A301-242A), SET1 (A300-290A), and NPAT (A302-772A) were purchased from Bethyl Laboratories. Anti-SMC3 antibody (sc-292645) was purchased from Santa Cruz Biotechnology. Anti-RAD21 antibody (05-908) was purchased from Millipore. Antibodies recognizing H3K4me3 (39915), H3K27ac (39133), and WDR5 (61485) were purchased from Active Motif. Antibodies recognizing CTCF (ab70303) and H3 (ab1791) were purchased from Abcam. Flag-HRP (A8592) and α-tubulin (T6199) antibodies were purchased from Sigma-Aldrich.
6
Western Blot Analysis of Protein Expression
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Cells lysate was prepared by using RIPA buffer followed with SDS-PAGE (Thermo Fisher Scientific) and transferred to a PVDF membrane according to the manufacturer’s protocols (Bio-Rad) at constant 100 V for 1 hr. After blocking incubation with 5% non-fat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 1 hr at room temperature, the membrane was incubated with antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), MYC (Cell Signaling Technology, #9402, 1:1,000), USF2 (Novus, NBP1-92649, 1:2,000), USF1 (Proteintech, 22327–1-AP, 1:2,000), GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), Vinculin (Proteintech, 26520–1-AP, 1:2,000) and CTCF (abcam, ab70303, 1:1,000) at 4°C for 12 hr with gentle shaking. Membranes were washed three times for 30 min and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 2 hr at room temperature. Blots were washed with TBS-T three times for 30 min and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocol.
7
Western Blot Analysis of CTCF, GAPDH, and Actin
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8
CTCF ChIP-qPCR in Fibroblasts
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ChIP was performed on chromatin isolated from patient-derived fibroblasts using the MAGnify Chromatin Immunoprecipitation System (Invitrogen) as per manufacturer recommendation. Chromatin was sonicated using the Covaris ME220 with the following parameters: PIP 75, DF 5%, CPB 200, 6°C setpoint for 16 min total, then precipitated using anti-CTCF (ab70303; Abcam) and rabbit IgG. To quantify CTCF occupancy, qPCR was performed with primers listed in Supplementary Table S1 using iTAQ SYBR (Bio-Rad) on the Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following conditions: 58°C annealing/extension. Results of the qPCR were analyzed using the ΔΔCT method (56 (link)).
9
Western Blot Analysis of Protein Biomarkers
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Protein lysates from tissues or cells were prepared in IP lysis buffer supplemented with protease inhibitor cocktail (Pierce, Thermo Fisher Scientific). Protein estimation was performed using the BCA Protein Assay Kit (Pierce). Equal amounts of protein lysates were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane for western blotting. Membranes were blocked with 5% non-fat milk, followed by incubation with primary antibody at 4°C overnight: anti-HOXA9 (ab140631, Abcam, Cambridge, UK), anti-β-catenin (71-2700, Invitrogen), anti-Twist (ab50581, Abcam), anti-E-cadherin (3195, Cell Signaling Technologies, Beverly, MA, USA), anti-N-cadherin (33-3900, Invitrogen), anti-Slug-1 (9585, Cell Signaling Technologies), anti-YAP1 (sc-15407, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HIF-1α (sc-10790, Santa Cruz), anti-CTCF (ab70303, Abcam), and anti-GAPDH (sc-25778, Santa Cruz). Membranes were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Bioworld Technology, Louis Park, MN, USA) at room temperature for 1 h. Western blots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Burlington, MA, USA) followed by film exposure. The relative expression of a target protein was normalized with internal control.
10
Immunoprecipitation of Nuclear Proteins
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Nuclei were isolated from MEL cells and lysed to make nuclear extracts. Nuclear extracts were then incubated with the indicated antibodies overnight at 4 °C. Protein G/A magnetic beads (Thermo Scientific) equilibrated with IP buffer (20 mM HEPES pH 7.9, 25% glycerol, 200 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.02 % NP-40) were added and the mixture was incubated for an additional 2 h at 4 °C. Beads were washed four times for 15 min and eluted by boiling in 2X Laemmli sample buffer.
The protein expression was detected as described66 . Briefly, cells were lysed in RIPA buffer (Boston Bioproducts) containing 1 mM DTT, 1 mM PMSF, and protease inhibitors and analyzed by SDS-PAGE and Western blotting using specific antibodies. For immunoprecipitation assays, protein extracts were mixed with Laemmli buffer and boiled at 95 °C for 5 min, followed by SDS-PAGE. Following antibodies diluted 1:1000 were used: Esco2 (bethyl, A301-689A), Matr3 (Abcam, ab84422), CTCF (Abcam, ab70303), Rad21 (Abcam, ab992), Smc3 (Abcam, ab9263), Mbd1 (Abcam, ab187734). Uncropped blots are provided in Source data.
The protein expression was detected as described66 . Briefly, cells were lysed in RIPA buffer (Boston Bioproducts) containing 1 mM DTT, 1 mM PMSF, and protease inhibitors and analyzed by SDS-PAGE and Western blotting using specific antibodies. For immunoprecipitation assays, protein extracts were mixed with Laemmli buffer and boiled at 95 °C for 5 min, followed by SDS-PAGE. Following antibodies diluted 1:1000 were used: Esco2 (bethyl, A301-689A), Matr3 (Abcam, ab84422), CTCF (Abcam, ab70303), Rad21 (Abcam, ab992), Smc3 (Abcam, ab9263), Mbd1 (Abcam, ab187734). Uncropped blots are provided in Source data.
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