Ab72418

Manufactured by Abcam

Sourced in United States

Ab72418 is a primary antibody used for the detection of target protein. It is designed for use in various immunoassay applications.

Automatically generated - may contain errors

Lab products found in correlation

Ab7028, by Abcam (2 mentions) Ab17721, by Abcam (2 mentions) Dynabeads protein g, by Thermo Fisher Scientific (2 mentions) Ab54583, by Abcam (2 mentions) Odyssey clx imager, by LI COR (1 mentions) Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions) Blocking buffer, by LI COR (1 mentions) Ab47383, by Abcam (1 mentions) P erk, by Cell Signaling Technology (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Ampure xp bead, by Illumina (1 mentions) Concanavalin a coated magnetic beads, by Bangs Laboratories (1 mentions) Odyssey infrared imager, by LI COR (1 mentions) Goat anti mouse irdye 680, by LI COR (1 mentions) Goat anti rabbit irdye 800, by LI COR (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions) Ab8106, by Abcam (1 mentions) Ab232820, by Abcam (1 mentions) Ab6276, by Abcam (1 mentions)

20 protocols using ab72418

Chd4 Interaction with Pdx1 in βTC3 Cells

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For co-immunoprecipitation experiments, 100 μg of βTC3 nuclear extract was incubated with either rabbit α-Chd4 (Abcam, ab72418; 2 μg) antibody or species-matched IgG overnight at 4°C. The following day, Protein A/G PLUS-agarose (Santa Cruz, sc-2003) was added for 2 hours at 4°C. Complexes were washed 5 times with PBS and boiled with SDS loading dye. Immunoprecipitates were fractionated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes, blocked with Blocking Buffer (LI-COR, 927-70001) and probed with the following primary antibodies: goat α-Pdx1 (Abcam, AB47383; 1:20000) and rabbit α-Chd4 (Abcam, ab72418, 1:500). Immunoblots were incubated with LI-COR IRDye secondary antibodies, and fluorometric scanning was performed with an Odyssey CLx Imager. Band intensity was quantitated using ImageJ software.

Davidson R.K., Weaver S.A., Casey N., Kanojia S., Hogarth E., Aguirre R.S., Sims E.K., Evans-Molina C, & Spaeth J.M. (2022). The Chd4 subunit of the NuRD complex regulates Pdx1-controlled genes involved in β-cell function. Journal of molecular endocrinology, 69(2), 329-341.

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Antibody Profiling for Cell Signaling

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The following antibodies were used in this study: CHD4 (ab72418, Abcam, polyclonal, dilution: 1:1000); PHF5A (ab103075; Abcam, polyclonal, dilution: 1:1000); myosin (MY-21, M4401, Sigma, monoclonal, dilution: 1:200); phospho–myosin (sc-12,896, Santa Cruz, monoclonal, dilution:1:200); ROCK (#4035S, Cell Signaling Technology, monoclonal, dilution:1:1000); RhoA (#2117, Cell Signaling Technology, monoclonal, dilution:1:1000). E-cadherin (#3195, Cell signaling Technology, monoclonal, dilution:1:1000); ERK (#4348S,Cell Signaling Technology, monoclonal, dilution:1:1000) and p-ERK (#4370, Cell Signaling Technology, monoclonal, dilution:1:2000).

Xu N., Liu F., Wu S., Ye M., Ge H., Zhang M., Song Y., Tong L., Zhou J, & Bai C. (2020). CHD4 mediates proliferation and migration of non-small cell lung cancer via the RhoA/ROCK pathway by regulating PHF5A. BMC Cancer, 20, 262.

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CHD4 Chromatin Profiling in Cells

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H9 cells and in vitro differentiated cells at different stages were harvested and gently homogenized with pestle A. Cells were then washed twice in 1.5 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 × Protease inhibitor cocktail, EDTA free), immobilized to concanavalin A coated magnetic beads (Bangs Laboratories), and then resuspended in 50 μl Dig-wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 × Protease inhibitor cocktail, 0.05% Digitonin) containing 2 mM EDTA^{85 (link)}. After sequential incubation with antibodies to CHD4 (Abcam, #ab72418, diluted 1:50 in 50 μl of Dig-Wash buffer), secondary antibodies (diluted 1:100 in 100 μl of Dig-Wash buffer) and a 1:200 dilution of pAG-Tn5 (gift from S. Henikoff) in Dig-300 Buffer (0.05% digitonin, 20 mM HEPES, pH7.5, 300 mM NaCl, 0.5 mM Spermidine, 1 × Protease inhibitor cocktail), bead-bound cells were resuspended in 50 μl tagmentation buffer (10 mM MgCl₂ in Dig-wash Buffer). The tagmented DNA was cleaned with 1.5 × Ampure XP beads (Beckman Counter), amplified with Illumina Nextera barcoded primers, and purified by 1.1 × Ampure XP beads for sequencing in a Novaseq 6000 instrument.

Lyu X., Rowley M.J., Kulik M.J., Dalton S, & Corces V.G. (2023). Regulation of CTCF loop formation during pancreatic cell differentiation. Nature Communications, 14, 6314.

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Keratinocyte Differentiation Protein Analysis

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Protein lysates from calcium-induced differentiated keratinocytes were obtained by scraping cells into a suitable amount of lysis buffer (50 mM Tris, pH 7.5, 150 mM sodium chloride, 5% glycerin, 1 mM DTT, 1 mM EDTA, 0.1 U/μl RiboLock RNase Inhibitor [Thermo Fisher Scientific], 1 mM AEBSF, 1x cOmplete Protease Inhibitor Cocktail [Roche]). Lysates were cleared for 15 min, at full speed at 4°C, and the protein concentration was determined via the Bradford assay. 30 μg of total protein was separated on a 10% SDS–PAGE and transferred onto the Amersham Hybond ECL membrane (Cytiva Life Science) by semi-dry blot. Blocking and antibody dilution were done in 5% milk powder in TBS-T. Primary antibodies included MTA2 (ab8106; Abcam) at 1:1,000 dilution, EGR3 (ab232820; Abcam) at 1:500 dilution, CHD4 (ab72418; Abcam) at 1:5,000 dilution, and β-actin (ab6276; Abcam) at 1:10,000 dilution. Secondary antibodies used were IRDye 800 goat anti-rabbit (926-32211; LI-COR Biosciences) and IRDye 680 goat anti-mouse (926-32220; LI-COR Biosciences), both at a 1:15,000 dilution. Western blots were analyzed with Odyssey Infrared Imager (LI-COR Biosciences).

Morgenstern E., Molthof C., Schwartz U., Graf J., Bruckmann A., Hombach S, & Kretz M. (2024). lncRNA LINC00941 modulates MTA2/NuRD occupancy to suppress premature human epidermal differentiation. Life Science Alliance, 7(7), e202302475.

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Proximity Ligation Assay for Protein Interactions

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PLA (Duolink; Olink Bioscience, Uppsala, Sweden) was performed according to the manufacturer's protocol with the following primary antibodies: mouse monoclonal anti-SCP3 (Abcam; AB97672) at a 1:100 dilution, rabbit anti SUMO-1 (Abcam; AB32058) at a 1:100 dilution, rabbit anti-phospho ATR (S428) (Cell Signaling Technology; 2 853) at a 1:100 dilution, sheep anti-Setx (in-house) at a 1:300 dilution, rabbit anti-CHD4 (Abcam; AB72418), and rabbit anti-BRCA1 (with courtesy of Prof. David Livingstone, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA) at a 1:300 dilution. Corresponding PLA PLUS and MINUS probes were subsequently applied. Slide mounting and imaging was performed as described above.

Yeo A.J., Becherel O.J., Luff J.E., Graham M.E., Richard D, & Lavin M.F. (2015). Senataxin controls meiotic silencing through ATR activation and chromatin remodeling. Cell Discovery, 1, 15025-.

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Co-immunoprecipitation of Lsd1, HDAC1, and Mi-2b

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Co-immunoprecipitation experiments was also performed as described (Whyte et al., 2012 (link)). Briefly, undifferentiated F9 ECCs and ESCs were washed and harvested in cold 1X PBS. Cellular proteins were extracted using TNEN250 lysis buffer (50 mM Tris pH 7.5, 5 mM EDTA, 250 mM NaCl, 0.1% NP-40) complemented with protease inhibitors at 4°C with rotation for 30 min. Complexes were then immunoprecipitated overnight at 4^°C with rotation by incubating the supernatant solution supplemented with two volumes of TNENG (50 mM Tris pH 7.5, 5 mM EDTA, 100 mM NaCl, 0.1% NP-40,10% glycerol) with Dynabeads® M280 (Life Technologies, 11203D) bound to 5 ug of antibody. Beads were washed withTNEN125 (50 mM Tris pH 7.5, 5 mM EDTA, 125 mM NaCl, 0.1% NP-40) and samples were eluted by boiling for 10 min in Laemmli’s loading buffer containing 100 mM DTT. Western blots were performed with NuPAGE4%–12% Tris-Bisgels. Antibodies used included: Lsd1 (abcam, ab17721), HDAC1 (abcam, ab7028), Mi-2b (abcam, ab72418).

AlAbdi L., Saha D., He M., Dar M.S., Utturkar S.M., Sudyanti P.A., McCune S., Spears B.H., Breedlove J.A., Lanman N.A, & Gowher H. (2020). Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers. Cell reports, 30(5), 1478-1490.e6.

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Co-IP and GST Pull-down Assays for Oct4 Interactions

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For co-immunoprecipitation (Co-IP), the nuclear extract was prepared according to manufacturer’s protocol (Active Motif, 40010) except that DNase was added for the release of chromatin-associated proteins. The Co-IP was performed using 1:1 mix of Dyna- beads Protein A (Life Technologies, 1002D) and Dyna-beads Protein G (Life Technologies, 1004D), and conjugated with 5 mg antibody and with 50 mg of nuclear extract according to the manufacturer’s protocol. Antibodies used include: anti-Oct4 (Abcam, ab181557), anti-CHD4 (Abcam, ab72418), anti-HDAC1 (Abcam, ab7028), anti-Lsd1 (Abcam, ab17721) and anti-HBO1 (Abcam, ab124993).
Pull down assays were performed using 1 mg of GST-Lsd1 (Sigma, SRP0122) incubated with 1 mg of recombinant Oct4 (abcam, ab134876) and Glutathione Sepharose 4B (GE healthcare, 17–0756-01) resin in the binding buffer (50 mM Tris pH 8.5, 50 mM KCl, 5 mM MgCl, 0.5% BSA, and 5% glycerol, complemented with a cocktail of protease inhibitors) overnight at 4°C with gentle agitation. The resin was washed twice with binding buffer and proteins were eluted using the elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8) according to the manufacturer’s instructions. Eluate and input were loaded onto a 10% SDS-PAGE gels and blots were probed using anti-Lsd1 (Abcam, ab17721) and anti-Oct4 (Santa Cruz, sc-8628) antibodies.

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Immunoprecipitation and Western Blot Analysis

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Cells were lysed with lysis buffer (25 mM Tris at pH 7.5, 10% glycerol, 150 mM NaCl, 1.5 mM MgCl₂, and protease inhibitors) containing 1% Triton X-100. The protein extracts were precleared with Dynabeads protein G (Invitrogen). The precleared extracts were incubated with the anti-Mi2β (Abcam, ab72418), JUNB (CST, C37F9), and cJUN (Abcam, ab31419) or the relevant isotype control in the presence of Dynabeads protein G and rotated overnight. Beads were then collected, washed at least five times with lysis buffer, and resuspended in SDS sample buffer. Eluents were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes, probed with anti-Mi2β (CST, 12011), JUNB (CST, C37F9), cJUN (BD Bioscience, 610327), HDAC2 (CST, D6S5P), and MTA3 (Bethyl Laboratories, A300-160), and examined by autoradiography by Enhance Chemical Luminescence.

Shibata S., Kashiwagi M., Morgan B.A, & Georgopoulos K. (2019). Functional interactions between Mi-2β and AP1 complexes control response and recovery from skin barrier disruption. The Journal of Experimental Medicine, 217(3), jem.20182402.

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ChIP-seq of Histone Modifications and Transcription Factors

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ChIP for histone modifications and transcription factors was performed from sorted WT and ΔIkE5^Cd2 large pre-B cells as described previously (Hu et al. 2016 (link)). In addition, ChIPs for Mi-2β and CTCF were performed with antibodies ab72418 (Abcam) and 2899s (Cell Signaling), respectively. DNA recovered from ChIP was used to generate libraries for sequencing. Briefly, 2.5–40 ng of DNA was end-repaired, end-adenylated, and then ligated with Illumina TruSeq-indexed adaptors. The ligated DNA was purified with AMPure XP beads (Beckman Coulter) and then amplified with KAPA HiFi DNA polymerase (KAPA Biosystems) for eight to 13 cycles. After amplification, the library DNA was separated on a 2% agarose gel, and DNA fragments in the 200- to 500-bp range were purified with a gel DNA recovery kit (Zymo Research). The purified DNA was diluted to 10 nM and multiplexed for sequencing at the Bauer Center Systems Biology Core at Harvard University. Image analysis and base calling were performed using the Illumina HiSeq 2000 software. Raw sequencing data sets were uploaded to DNAnexus, a cloud-based genome informatics and data management platform.

Yoshida T., Hu Y., Zhang Z., Emmanuel A.O., Galani K., Muhire B., Snippert H.J., Williams C.J., Tolstorukov M.Y., Gounari F, & Georgopoulos K. (2019). Chromatin restriction by the nucleosome remodeler Mi-2β and functional interplay with lineage-specific transcription regulators control B-cell differentiation. Genes & Development, 33(13-14), 763-781.

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Immunofluorescence Assay of Murine Lung Development

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Lungs from wild type mouse embryos from E11 till E18 were dissected and fixed in 4% PFA overnight at 4°C before processing for paraffin embedding. Sections of 5 μm were dewaxed and rehydrated, followed by antigen retrieval with microwave treatment in TE. Samples were blocked and incubated with antibodies against CHD4 (ab72418) and SOX2 (GTI5098), FOXP2 (ab16046) and SOX2, and CUX1 (Abcam, Ab54583) and SOX2 diluted in blocking buffer at 4°C overnight. After washing in PBS with 0.05% Triton X-100, slides were incubated in blocking buffer with fluorophore conjugated secondary antibody (Alexa Fluor-488, Alexa Fluor-594; Jackson Immuno Research) for 1 h at room temperature. Slides were mounted in Vectashield Mounting Medium with Dapi (Vector laboratories, Burlingame, CA, United States). Digital images were captured using a ZEISS imager Z1 AX10 microscope.

Schilders K.A., Edel G.G., Eenjes E., Oresta B., Birkhoff J., Boerema-de Munck A., Buscop-van Kempen M., Liakopoulos P., Kolovos P., Demmers J.A., Poot R., Wijnen R.M., Tibboel D, & Rottier R.J. (2022). Identification of SOX2 Interacting Proteins in the Developing Mouse Lung With Potential Implications for Congenital Diaphragmatic Hernia. Frontiers in Pediatrics, 10, 881287.

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