The following antibodies were used as primary antibodies for immunofluorescence microscopy: SGO1 (SAB1405371, Sigma Aldrich), GFP (ab290, Abcam) and CENPA (07–574, Millipore; and ab13939, Abcam). For immunoblotting, the following primary antibodies were used: SA1 (ab4457, Abcam), SA2 (A300-158a, Bethyl Laboratories), SMC1 (A300-055A, Bethyl Laboratories), SCC1 (05-908, Millipore), WAPL (A-7, sc-365189, Santa Cruz), Sororin (ab192237, Abcam), HSP90 (sc-13119(F-8), Santa Cruz) and α-tubulin (T5168, Sigma Aldrich). All primary antibodies were used at a 1:1000 dilution with the exception of HSP90 and α-tubulin (1:10000). For coimmunoprecipitation, we used 4.5 μg of SMC1 (A300-055A, Bethyl Laboratories) or IgG (2729 S, Cell Signaling) per sample. Secondary antibodies were used at a 1:1000 dilution. For immunofluorescence microscopy we used: Alexa Fluor 488 goat anti-mouse (A-11001, Life Technology), Alexa Fluor 568 goat anti-mouse (A-11004, Life Technology), Alexa Fluor 488 goat anti-rabbit (A-11008, Life Technology) and Alexa Fluor 568 goat anti-rabbit (A-11011, Life Technology). For western blots, we used the following secondary antibodies: anti-goat-PO (P0449, DAKO), anti-rabbit-PO (P0448, DAKO) and anti-mouse-PO (P0447, DAKO).
Ab4457
Manufactured by Abcam
Ab4457 is a laboratory equipment product manufactured by Abcam. It is a device used for a specific function in scientific research and experimentation, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Sc 365189, by Santa Cruz Biotechnology (1 mentions)
P0448, by Agilent Technologies (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Ab13939, by Abcam (1 mentions)
Ab192237, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Ab290, by Abcam (1 mentions)
P0449, by Agilent Technologies (1 mentions)
Ab6002, by Abcam (1 mentions)
Truseq indexed adapter, by Illumina (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Kapa hifi dna polymerase, by Roche (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Na934, by GE Healthcare (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
A5316, by Merck Group (1 mentions)
Nxa931, by GE Healthcare (1 mentions)
5 protocols using ab4457
1
Antibody Characterization for IF and IB
2
Chromatin Immunoprecipitation Sequencing Protocol
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ChIP assays were performed as previously described.[20 (link), 25 (link), 26 (link)] Samples were immunoprecipitated with antibody against CTCF (Creative Diagnostics, DMABT-H19813), the SA-1 subunit of cohesin (Abcam ab4457), trimethyl histone H3 lysine 27 (Abcam ab6002) or nonspecific rabbit IgG (sc-2091 Santa Cruz). DNA processing and high throughput sequencing were performed as described.[20 (link)] Because of the age, gender, and genetic background differences noted above, and the growing realization genetic variability influences epigenetic findings, [27 (link)] parallel RNA-seq and ChIP-seq of CTCF and cohesinSA1 data sets from individual donors were analyzed together.
3
ChIP-seq Protocol for Chromatin Binding Factors
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Cells were fixed in medium or phosphate buffered saline with 1% formaldehyde at room temperature for 10 min. ChIP experiments were performed as described46 (link). ChIP-seq libraries were prepared using NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (New England BioLabs, E6240). Rabbit polyclonal antibody for Rad21 (1:1000 dilution for western blot; 2.5 ug/million cells for ChIP-seq) was obtained from Eurofins Genomics and has been described in47 (link). Antibodies for MAU2 (ab46906, 2.5 ug/million cells as dilution) and SA1 (ab4457, 2.5 ug/million cells) were from Abcam. Antibodies for TAF1 (A303-505A, 2.5 ug/million cells) and AFF4 (A302-538A, 2.5 ug/million cells) were from Bethyl Laboratory. CTCF (07-729, 2.5 ug/million cells) antibody was from Merck Millipore. Antibodies (2.5 ug/million cells) for unphosphorylated Pol2 (CMA601), Pol2ser2 (CMA602) and H3K27ac (CMA309) were kindly provided by Dr. H Kimura (TITech), which were used in previous studies17 (link),48 (link). Antibody for CBP (606402, 2.5 ug/million cells) was from BioLegend. Antibodies for P300 (sc-585, 2.5 ug/million cells) and Med1 (sc-5334, 2.5 ug/million cells) were from Santa Cruz Biotechnology.
4
ChIP-seq Protocol for Chromatin Profiling
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ChIP-seq experiments were performed as previously described40 (link). Briefly, cells were fixed with 1% formaldehyde at RT for 10 min, lysed and sonicated in RIPA buffer containing 0.2% SDS (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.2% SDS, 0.5 mM PMSF, and 1x Protease inhibitor cocktail (Roche). Chromatin was cleared by centrifugation at 20,000×g for 10 min and incubated with 2–10 μg of antibodies pre-bound to Dynabeads Protein G (Life technologies). The antibodies used here were specific for STAG1 (Abcam, ab4457, lot: GR279696-4), STAG2 (Abcam, ab4464, Lot: GR271549-1), SMC1A (Bethyl, A300-055A, lot 5), and CTCF (Cell Signaling Tech, 2899 s, lot 2). Purified ChIP DNA was end-repaired, end adenylated, and ligated with Illumina Truseq indexed adapters. The ligated DNA was purified with AMPure XP beads (Beckman Coulter) and amplified with KAPA HiFi DNA Polymerase (KAPA Biosystems) for 8 to 13 cycles. After amplification, the library DNA was size-selected with AMPurex XP beads to 200–600 bp range, and the purified libraries were multiplexed for sequencing at the Center for Computational and Integrative Biology (CCIB) DNA Core at MGH.
5
Western Blot Analysis of Chromatin Proteins
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Cells were trypsinized, counted, washed with ice-cold PBS and lysed in Laemmli buffer (50 mM Tris-HCL, 2.5 mM EDTA, 2.55 mM EGTA, 2% SDS 20%, 5% Glycerol, 1% Bromophenol blue, protease inhibitor cocktail tablets and 2 mM DL-Dithiothreitol solution) at 10 million cells/ml. Protein lysates were sonicated and denatured at 95 C for 5 min and electrophorated on 4-15% Mini-PRO-TEANÒTGXTM gels (456-1084, BIO-RAD), transferred onto nitrocellulose membranes (1704159, BIO-RAD). Membranes were incubated overnight at 4 C with mouse anti-STAG2 (1:1,000, Santa Cruz Biotechnology, sc-81852), goat anti-STAG1 (1:5,000, ab4457, Abcam), rabbit anti-H3 (1:50,000, ab1791, Abcam), mouse anti-b-Actin (1:20,000, A5316, Sigma-Aldrich), rabbit anti-FLI1 antibody (1:1,000, ab133485, Abcam) and rabbit anti-H3K27ac (1:1,000, ab4729, Abcam). Then membranes were incubated 1h at room temperature with respective anti-rabbit, anti-mouse immunoglobulin G horseradish peroxidase (HRP) coupled secondary antibody (1:3,000, NA934 or NXA931, respectively; GE Healthcare) or anti-goat IgG-HRP (1: 10,000, SC-2354, Santa Cruz). Proteins were visualized using SuperSignalÔ West Pico Plus (34580, Thermo Scientific) and ChemiDocÔ Imaging System (BIO-RAD).
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