Ab5089

Manufactured by Abcam

Sourced in United Kingdom

Ab5089 is a recombinant monoclonal antibody, which is designed for use as a research tool. The core function of this product is to serve as a detection reagent for target proteins in various applications, such as Western blotting, immunohistochemistry, and flow cytometry.

Automatically generated - may contain errors

Lab products found in correlation

Ab9110, by Abcam (3 mentions) Nextseq 500, by Illumina (2 mentions) H3k27ac antibody, by Diagenode (2 mentions) Sc 543, by Santa Cruz Biotechnology (2 mentions) Ab4729, by Abcam (2 mentions) Ab93806, by Abcam (2 mentions) Dynabeads protein a, by Thermo Fisher Scientific (1 mentions) Ab24551, by Abcam (1 mentions) Pg 21, by Merck Group (1 mentions) Pa1 844a, by Thermo Fisher Scientific (1 mentions) Ab23738, by Abcam (1 mentions) Hiseq 2000, by Illumina (1 mentions) Streptavidin hrp, by Jackson ImmunoResearch (1 mentions) Anti myc 2276, by Cell Signaling Technology (1 mentions) Anti gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions) Anti flag f1804, by Merck Group (1 mentions) Anti erα sc 543, by Santa Cruz Biotechnology (1 mentions) Anti yap1 14074s, by Cell Signaling Technology (1 mentions) Anti histone h3, by GenScript (1 mentions) Ab46540, by Abcam (1 mentions)

14 protocols using ab5089

Protein Isolation and Co-immunoprecipitation Protocols

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Proteins for Western blotting were isolated using TIVE lysis buffer (50 mM Tris-HCl pH 7.8, 2 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitors), or lysis buffer A (10 mM HEPES pH 7.5, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, protease inhibitors) plus 0.5% NP-40 and lysis buffer C (20 mM HEPES pH 7.5, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% Glycerol, 1 mM DTT, protease inhibitors) for total, cytoplasmic and nuclear proteins respectively. Co-immunoprecipitation (co-IP) was carried using the nuclear protein fraction, similarly to previously described (Jehle et al., 2014 (link)) and ARv7 antibody-coupled Protein A Dynabeads (Thermo Fisher Scientific). Co-IP experiments with formaldehyde crosslinked material was carried out using isolated chromatin samples, as described below. Western blotting was carried out using following antibodies: AR (N20 and 441, Santa-Cruz Biotechnologies; PG21, Millipore), AR C-terminus (SP242, Spring Bioscience; C-19, Santa-Cruz Biotechnologies), ARv7 (RM7, RevMAb; H6 253, Epitomics), NCOR1 (PA1–844A, Invitrogen), NCOR2 (ab24551, Abcam), FOXA1 (ab5089, Abcam), b-Actin (Abcam) and Lamin B1 (EPR8985, Abcam), and secondary antibody IgG fraction monoclonal mouse anti-rabbit IgG, light chain specific (211–032-171, Jackson ImmunoResearch).

Cato L., de Tribolet-Hardy J., Lee I., Rottenberg J.T., Coleman I., Melchers D., Houtman R., Xiao T., Li W., Uo T., Sun S., Kuznik N.C., Gö ppert B., Ozgun F., van Royen M.E., Houtsmuller A.B., Vadhi R., Rao P.K., Li L., Balk S.P., Den R.B., Trock B.J., Karnes R.J., Jenkins R.B., Klein E.A., Davicioni E., Gruhl F.J., Long H.W., Liu X.S., Cato A.C., Lack N.A., Nelson P.S., Plymate S.R., Groner A.C, & Brown M. (2019). ARv7 Represses Tumor-Suppressor Genes in Castration-Resistant Prostate Cancer. Cancer cell, 35(3), 401-413.e6.

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Genome-wide ChIP-seq of ER, FOXA1, GATA3 and H3K27Ac

Cited 1 time

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ChIP experiments were conducted as described previously (16 (link)) and were done in duplicates. MCF7, T47D, MDAMB134 and SUM44 cells were cultured for three days in HD conditions then treated with 10nM estradiol for 45 minutes. For the ChIP experiments with tamoxifen we used 10nM 4-hydroxytamoxifen for 45 minutes. Chromatin from 20 million formaldehyde-fixed cells was sonicated to a size range of 200–300 bp. Solubilized chromatin was immunoprecipitated with a mix of the ER antibodies Ab10 (Thermo Fisher Scientific) and SC-543 (Santa Cruz), a mix of the FOXA1 antibodies ab5089 and ab23738 (Abcam), GATA3 antibody D13C9 (Cell signaling) or H3K27Ac antibody (C15410196, Diagenode). The same antibodies were used in each experiment for all the cell lines. The samples were reversed crosslinked, treated with proteinase K, and DNA was extracted. Libraries were sequenced using 75 bp paired-end reads on the Illumina Nextseq500 at the Dana-Farber Cancer Institute.

Nardone A., Qiu X., Spisak S., Nagy Z., Feiglin A., Feit A., Cohen Feit G., Xie Y., Font-Tello A., Guarducci C., Hermida-Prado F., Syamala S., Lim K., Munoz Gomez M., Pun M., Cornwell M., Liu W., Ors A., Mohammed H., Cejas P., Brock J.B., Freedman M.L., Winer E.P., Fu X., Schiff R., Long H.W., Metzger Filho O, & Jeselsohn R. (2022). A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer. Cancer research, 82(20), 3673-3686.

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ChIP-seq protocol for liver tissue

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The ChIP-seq protocol used was as described by Schmidt et al.^{68 (link)} Briefly, livers were isolated from 10 to 12 weeks old mice and liver tissue was post mortem cross-linked using 1% formaldehyde (v/v), lysed and sonicated. Protein-bound DNA was immunoprecipitated using 10 µg of an antibody against CEBPA (Santa Cruz, sc-9314), HNF4A (ARP 31946_P050), FOXA1 (ab5089, Abcam), H3K27ac (ab4729, Abcam), or H3K4me3 (Millipore 05-1339). Immunoprecipitated DNA was end-repaired at 20 °C for 30 min, Adenine overhang was added at 37 °C for 30 min, and Illumina sequencing adapters ligated at room temperature for 15 min before 16 cycles of PCR amplification. PCR conditions: (1) 98 °C—30 s; (2) 98 °C—30 s, 65 °C—30 s, 72 °C—30 s, 16 cycles; (3) 72 °C—5 min. DNA fragments ranging from 200- to 300-bp in size were selected on a 2% agarose gel for 50-bp single-end read sequencing on an Illumina HiSeq 2000 according to the manufacturer’s instructions.

Wong E.S., Schmitt B.M., Kazachenka A., Thybert D., Redmond A., Connor F., Rayner T.F., Feig C., Ferguson-Smith A.C., Marioni J.C., Odom D.T, & Flicek P. (2017). Interplay of cis and trans mechanisms driving transcription factor binding and gene expression evolution. Nature Communications, 8, 1092.

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Protein Interaction Mapping by BioID

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Whole cell lysates or cytoplasmic/nuclear fractions were extracted for Western blotting assays. Proteins were separated by SDS-PAGE gel and identified by antibodies. The antibodies used in this assay were: anti-ERα (sc-543, Santa Cruz), anti-YAP1 (14074S, Cell Signaling Technology), anti-TEAD4 (sc-101184, Santa Cruz), anti-Histone H3 (A01502, GenScript), antiHA (ab9110, Abcam), anti-Myc (2276S, Cell Signaling Technology), anti-FOXA1 (ab5089, Abcam), anti-TAZ (sc-293183, Santa Cruz), anti-Flag (F1804, Sigma) and anti-GAPDH (sc-25778, Santa Cruz). For BioID system, the in vivo proximity biotinylation events mediated by BioID-tagged ERα or FOXA1 can be detected by Western blot using streptavidin-HRP (016-030-084, Jackson ImmunoResearch).

Zhu C., Li L., Zhang Z., Bi M., Wang H., Su W., Hernandez K., Liu P., Chen J., Chen M., Huang T.H., Chen L, & Liu Z. (2019). A non-canonical role of YAP/TEAD is required for activation of estrogen-regulated enhancers in breast cancer. Molecular cell, 75(4), 791-806.e8.

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Chromatin Immunoprecipitation Assay in MCF-7 Cells

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MCF-7 cells were grown for 2 days in HD medium before siRNA transfection or 17β-estradiol (E2) treatment. ChIP experiments were performed as described in [4 (link)]. qRT-PCR was carried out on ChIP-enriched DNA using SYBR Green Master Mix. ChIP enrichment was normalized on input samples (1% of total chromatin used per IP) and expressed as enrichment of specific binding over the control unspecific IgG binding. Antibodies against human ERα (Santa Cruz Biotechnology; sc534X, sc7207X), AP-2γ (Santa Cruz Biotechnology; sc-8977X), FoxA1 (Abcam; ab5089) and IgG (Abcam, ab46540) were used in this assay. Custom ChIP-primer pairs are reported in Supplemental Table 6.

Miano V., Ferrero G., Reineri S., Caizzi L., Annaratone L., Ricci L., Cutrupi S., Castellano I., Cordero F, & De Bortoli M. (2015). Luminal long non-coding RNAs regulated by estrogen receptor alpha in a ligand-independent manner show functional roles in breast cancer. Oncotarget, 7(3), 3201-3216.

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Immunoblotting Protein Expression Analysis

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Cells were grown to 60 to 70% confluence and lysed with standard radioimmunoprecipitation assay buffer. The resulting total cell lysate was run on SDS–polyacrylamide gel electrophoresis gel, transferred onto nitrocellulose membrane, and immunoblotted using antibodies for the proteins of interest. Antibodies used for immunoblotting are anti-ER (HC-20 from Santa Cruz Biotechnology), anti-PR KD68 (in-house developed), anti-actin (A2228 from Sigma), anti-FOXA1 (Ab5089 from Abcam), and anti-NF1C (gift from N. Tanese, New York University). Protein expression was normalized to actin loading control.

Singhal H., Greene M.E., Tarulli G., Zarnke A.L., Bourgo R.J., Laine M., Chang Y.F., Ma S., Dembo A.G., Raj G.V., Hickey T.E., Tilley W.D, & Greene G.L. (2016). Genomic agonism and phenotypic antagonism between estrogen and progesterone receptors in breast cancer. Science Advances, 2(6), e1501924.

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ChIP-seq protocol for epigenetic profiling

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ChIP experiments were conducted as described previously (Wang et al., 2009 (link)) and were done in triplicates. Chromatin from approximately 1 × 10⁷ fixed cells was sonicated to a size range of 200-300 bp. Solubilized chromatin was subjected to immunoprecipitation with the ER antibody, Ab10 (Lab Vision corporation) and SC-543 (Santa cruz), HA antibody (Ab9110, Abcam), H3K27Ac antibody (C15410196, Diagenode) or FOXA1 (ab5089,Abcam) bound to protein A and protein G beads (Life Technologies). A fraction of the sample was not exposed to antibody to be used as control (input). The samples were reversed crosslinked, treated with proteinase K, and DNA was extracted. DNA sequencing libraries were prepared using the ThruPLEX-FD Prep Kit (Rubicon Genomics). Libraries were sequenced using 50 bp reads on the Illumina Nextseq500 at the Dana-Farber Cancer Institute.

Jeselsohn R., Bergholz J.S., Pun M., Cornwell M., Liu W., Nardone A., Xiao T., Li W., Qiu X., Buchwalter G., Feiglin A., Abell-Hart K., Fei T., Rao P., Long H., Kwiatkowski N., Zhang T., Gray N., Melchers D., Houtman R., Liu X.S., Cohen O., Wagle N., Winer E.P., Zhao J, & Brown M. (2018). Allele-specific chromatin recruitment and therapeutic vulnerabilities of ESR1 activating mutations. Cancer cell, 33(2), 173-186.e5.

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Western Blot Analysis of Key Proteins

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Total proteins were extracted from cells using Laemmli lysis buffer, supplemented with a complete protease inhibitor cocktail (Roche). Per sample, 40 μg of protein was resolved by SDS-PAGE (10%) and transferred on nitrocellulose membranes (Santa Cruz Biotechnology). The following antibodies were used for Western blot stainings: ARNTL (ab93806, Abcam), PSA (5365, Cell Signaling Technology), FOXA1 (ab5089, Abcam), GR (12041, Cell Signaling Technology) and ACTIN (MAB1501R, Merck Millipore). Blots were incubated overnight at 4°C with designated primary antibodies at 1:1000 (ARNTL, PSA) or 1:5000 (ACTIN) dilution, and visualized using the Odyssey system (Li-Cor Biosciences).

Linder S., Hoogstraat M., Stelloo S., Eickhoff N., Schuurman K., de Barros H., Alkemade M., Bekers E.M., Severson T.M., Sanders J., Huang C.C., Morova T., Altintas U.B., Hoekman L., Kim Y., Baca S.C., Sjöström M., Zaalberg A., Hintzen D.C., de Jong J., Kluin R.J., de Rink I., Giambartolomei C., Seo J.H., Pasaniuc B., Altelaar M., Medema R.H., Feng F.Y., Zoubeidi A., Freedman M.L., Wessels L.F., Butler L.M., Lack N.A., van der Poel H., Bergman A.M, & Zwart W. (2022). Drug-induced epigenomic plasticity reprograms circadian rhythm regulation to drive prostate cancer towards androgen-independence. Cancer discovery, 12(9), 2074-2097.

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Chromatin Fractionation and Western Blot

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Cells were washed twice in phosphate buffered saline (1XPBS) and cell pellet was carefully suspended in 200ul of SFI Buffer (100mM NaCl, 300mM Sucrose, 3mM MgCl2, 10mM PIPES [pH 6.8], 1mM EGTA, 0.2% Triton X-100,) with PIC. Cells were incubated for 30 mins at 4°C and then pelleted down at 2900 rpm for 5 min at 4°C, and the supernatant was collected (Soluble fraction). The pellet was carefully washed twice in SFI buffer and chromatin bound fraction was extracted by adding 2X protein loading dye to the pellet. Fractions were loaded on 15% SDS PAGE and western was done for ERα (sc-8002 Santa Cruz Biotechnology), GAPDH (sc-32233 Santa Cruz Biotechnology), Histone H3 (H0164, Sigma-Alderich). Cellular lysates prepared were separated on 15% SDS-PAGE gels. The protein transfer was carried out in Tris-glycine buffer at 30V for 1 hour on ice using 0.45μ PVDF membrane (Millipore). Membrane was blocked in 5% non-fat dry milk made in TBST and further incubated for three hours with primary antibodies followed by stringent washings. Membranes were probed with HRP-conjugated secondary antibody (Bio-rad Laboratories). Signal amplification was performed using ECL substrate (RPN2106, GE Healthcare). Images were captured on Image Quant LAS4000 with CCD camera. Anti-FOXA1 (ab5089, Abcam) and MED1 (A3007938, Bethyl) were used for total protein levels.

Saravanan B., Soota D., Islam Z., Majumdar S., Mann R., Meel S., Farooq U., Walavalkar K., Gayen S., Singh A.K., Hannenhalli S, & Notani D. (2020). Ligand dependent gene regulation by transient ERα clustered enhancers. PLoS Genetics, 16(1), e1008516.

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Sequential ChIP-re-ChIP Analysis

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The ChIP-re-ChIP assay was performed with Re-ChIP-IT kit according to manufacturer’s instructions (Active Motif, 53016). Anti-HNF4G (Proteintech; 25801-1-AP) and anti-FOXA1 (Abcam; ab5089) were used for the first ChIP; eluates from FOXA1 ChIP were used to perform 2^nd ChIP with same anti-HNF4G, anti-FOXA1 or no antibody control.

Shukla S., Cyrta J., Murphy D.A., Walczak E.G., Ran L., Agrawal P., Xie Y., Chen Y., Wang S., Zhan Y., Li D., Wong W.P., Sboner A., Beltran H., Mosquera J.M., Sher J., Cao Z., Wongvipat J., Koche R.P., Gopalan A., Zheng D., Rubin M.A., Scher H.I., Chi P, & Chen Y. (2017). Aberrant activation of a gastrointestinal transcriptional circuit in prostate cancer mediates castration resistance. Cancer cell, 32(6), 792-806.e7.

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