Dynabeads protein g

Manufactured by Abcam

Sourced in Australia, United Kingdom, United States

Dynabeads® protein G are superparamagnetic beads coated with recombinant protein G. Protein G is a bacterial cell wall protein that binds to the Fc region of various immunoglobulin classes. These beads can be used for the rapid and efficient isolation and purification of antibodies from different sample sources.

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Lab products found in correlation

Ip lysis buffer, by Thermo Fisher Scientific (3 mentions) Protein g dynabead, by Thermo Fisher Scientific (3 mentions) Dynabeads protein g, by Thermo Fisher Scientific (3 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Ab290, by Abcam (2 mentions) Pms2 gfp, by Addgene (2 mentions) Hiseq 4000 platform, by Illumina (2 mentions) Pcr purification kit, by Qiagen (2 mentions) Anti yap1 antibody, by Abcam (1 mentions) Ab70892, by Abcam (1 mentions) Ne per nuclear cytoplasmic extraction reagents, by Thermo Fisher Scientific (1 mentions) Glycine, by Merck Group (1 mentions) Ez magna chip kit, by Merck Group (1 mentions) Ab31419, by Abcam (1 mentions) Ab72418, by Abcam (1 mentions) Ripa buffer, by Beyotime (1 mentions) Cp750, by Cole-Parmer (1 mentions) Anti h3k27ac antibody, by Abcam (1 mentions) Anti bax, by Abcam (1 mentions) Anti p53, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Dynabeads (17 citations) Dynabeads™ Protein (2 citations) Dynabeads® protein G beads (2 citations)

15 protocols using dynabeads protein g

Investigating Protein Interactions via Co-IP Assay

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Co-IP assay was performed to assess the interactions between proteins. In detail, U251 cells were lysed in IP lysis buffer (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. Next, the cell lysate containing 200 μg proteins was incubated with Dynabeads^® protein G for 1 hr, and incubated with 2 μg anti-YAP1 antibody (No. ab56701; Abcam) or IgG (negative control) overnight at 4°C, followed by incubation with Dynabeads^® protein G for another 1 hr. Then, the immune complex was submitted to western blotting assay with antibodies against Ub (No. 3933), TEA/ATTS domain (TEAD , No. 13295), P73 (No. 14620) and runt-related
transcription factor 2 (RUNX2; No. 12556), all purchased from Cell Signaling Technology, and then anti-cAMP responsive
element-binding protein 1 (CREB; No. ab31387; Abcam) antibody.

Xu X., Chen Y., Wang X, & Mu X. (2019). Role of Hippo/YAP signaling in irradiation-induced glioma cell apoptosis. Cancer Management and Research, 11, 7577-7585.

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MS2-GFP-RIP Assay for ERG CDS Regions

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MS2-GFP-RIP assays with the CDS regions of 3 ERGs fused with MSbs sequences were performed as previously described^{67 (link)}. In details, the empty plasmid pcDNA3.1-MS2 or the plasmid with the selected CDS regions inserted was co-transfected with pMS2-GFP (Addgene) into HEK293FT cells. Forty-eight hours after transfection, cells (10 million) were harvested and lysed with 1 ml native lysis buffer (50 mM Tris pH7.4, 150 mM NaCl, 0.5% NP-40, 0.5 mM PMSF, 2 mM RVC, protease inhibitor cocktail (Roche)) followed by sonication. The lysate was centrifuged at 10,000 × g for 30 min at 4 °C, and the supernatant was pre-cleared with 10ul Dynabeads Protein-G. The samples were then incubated with GFP antibody (3 µg per reaction; ab290, abcam) for 2 h at 4 °C, followed by addition of 20 μl Dynabeads Protein-G to the mixture and incubation overnight at 4 °C on a rotating shaker. Next, the samples were washed with wash buffer (50 mM Tris pH7.4, 300 mM NaCl, 0.5% NP-40, 0.5 mM PMSF, 2 mM RVC, protease inhibitor cocktail (Roche, 4693124001)) for 4 times at 4 °C. Finally, the magnetic beads were resuspended in 20 μl 1× SDS loading buffer and boiled for 10 min. The samples were analyzed by 10% SDS-PAGE and probed by immunoblotting with anti-eIF4A1 (1:1000, orb353605, Biorbyt).

Li F., Fang J., Yu Y., Hao S., Zou Q., Zeng Q, & Yang X. (2023). Reanalysis of ribosome profiling datasets reveals a function of rocaglamide A in perturbing the dynamics of translation elongation via eIF4A. Nature Communications, 14, 553.

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Eomes ChIP-seq in Embryoid Bodies

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Day 4 Eomes^V5/V5 (CCE) and WT (CCE) D4 EBs were dissociated and ~3-4x10^{7 (link)} cells were cross-linked for 10 min at RT with 1% (v/v) formaldehyde, and quenched with 125mM glycine. Nuclei were recovered and lysed to obtain chromatin, which was then sonicated to 200–500 bp, pre-cleared with protein G Dynabeads (Thermofisher Scientific) and ~175 ug of chromatin was immunoprecipitated with 10μg of anti-V5 (Abcam cat# ab9116, lot# GR: 322448-4) bound to protein G Dynabeads overnight on a rotator at 4°C. Dynabeads were washed and immune complexes were eluted in IP elution buffer (1% SDS, 0.1M NaHCO3), reverse crosslinked overnight at 65°C, RNaseA treated for 1.5hrs at 42°C, proteinase K treated for 2hrs at 45°C and DNA was recovered using a Zymo column kit. DNA was multiplexed and paired end sequencing was performed on a single lane of an Illumina HiSeq4000 platform.

Harland L.T., Simon C.S., Senft A.D., Costello I., Greder L., Imaz-Rosshandler I., Gottgens B., Marioni J.C., Bikoff E.K., Porcher C., de Bruijn M, & Robertson E.J. (2021). The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk-Sac Mesodermal Progenitors. Nature cell biology, 23(1), 61-74.

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Nuclear Protein Immunoprecipitation and Analysis

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One milligram of nuclear extracts was diluted in dilution buffer (50 mM Tris, pH 7.5, 0.3% NP-40, protease inhibitor, 1 mM DTT) to attain a final concentration of 150 mM NaCl. Nuclear extracts were pre-cleared for 3 h at 4°C by incubation with normal IgG (goat sc-2028 and rabbit sc-2027; Santa Cruz) and protein G Dynabeads (Life Technologies), blocked with 0.2 mg ml⁻¹ BSA (Pierce) and 0.4 mg ml⁻¹ sonicated salmon sperm DNA (Life Technologies). Immunoprecipitations were performed overnight at 4°C by incubation of the pre-cleared nuclear extracts with primary antibodies (for JARID1A, ab 70892; Abcam) and blocked protein G Dynabeads in dilution buffer (50 mM Tris, pH 7.5, 0.3% NP-40, protease inhibitor, 1 mM DTT). Beads were washed five times with 4× bed volume of wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.3% NP-40, protease inhibitor, 1 mM DTT) and bound material was eluted in 1× Laemmli buffer by boiling at 95°C for 10 min. Fractions were analysed by immunoblotting as described above.

Karia D., Gilbert R.C., Biasutto A.J., Porcher C, & Mancini E.J. (2020). The histone H3K4 demethylase JARID1A directly interacts with haematopoietic transcription factor GATA1 in erythroid cells through its second PHD domain. Royal Society Open Science, 7(1), 191048.

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Chromatin Immunoprecipitation for BRD4 Occupancy

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Nuclear fractions were extracted from cell pellets per protocol (NE-PER Nuclear & Cytoplasmic Extraction Reagents, Thermo Scientific), fixed in 16% formaldehyde, and chromatin fragmented by sonication. Diluted chromatin was immunoprecipitated using isotype control (IgG) or anti-BrD4 antibodies (Abcam) and Protein G Dynabeads. Following reversal of crosslinks and RNaseA/proteinase K digestion, immunoprecipitated DNA was purified using PCR purification kit, quantified by Nanodrop, and c-myc transcripts quantified by qPCR.

Murga-Zamalloa C., Polk A., Hanel W., Chowdhury P., Brown N., Hristov A.C., Bailey N.G., Wang T., Phillips T., Devata S., Poonnen P., Gomez-Gelvez J., Inamdar K.V, & Wilcox R.A. (2017). Polo-like-kinase 1 (PLK-1) and c-myc inhibition with the dual kinase-bromodomain inhibitor volasertib in aggressive lymphomas. Oncotarget, 8(70), 114474-114480.

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Chromatin Immunoprecipitation (ChIP) Protocol

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ChIP was performed, as previously described.^{9 (link)} Cells were cross-linked with 1% formaldehyde for 10 min and quenched with 125 mM glycine (Sigma-Aldrich) for 5 min. Cross-linked samples were lysed at 4 °C for 10 min and sonicated to generate chromatin fragments with an average size of 200–800 bp. Chromatin from 1 × 10⁶ cells was immunoprecipitated with Protein G Dynabeads and anti-RNApII (clone 4H8) and anti-Ser2 RNApII antibodies (both from Abcam, Waterloo, NSW, Australia). Cross-links in immunoprecipitated samples were reversed by incubating with 200 mM NaCl and 10 mg/ml of Proteinase K at 65 °C for 16 h and purified using the Qiagen PCR purification kit. Samples were analysed using qPCR with primers described in Supplementary Table SI.

Lee W., Johnson J., Gough D.J., Donoghue J., Cagnone G.L., Vaghjiani V., Brown K.A., Johns T.G, & St. John J.C. (2015). Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny. Cell Death & Disease, 6(2), e1664-.

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Eomes ChIP-seq in Embryoid Bodies

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ChIP Assay for SND1 Protein

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ChIP analysis was carried out using a Millipore EZ-Magna ChIP kit (cat. no. 17-295; EMD Millipore) according to the manufacturer's protocol. Briefly, the T24/R and 5637/R cells were seeded into a 6-well plate at a density of 5×10⁶ cells/well. The cells were collected after 24 h of treatment and fixed with 1% formaldehyde for 10 min at room temperature. After rehydrating in 0.125 M glycine, the cells were washed in phosphate-buffered saline (PBS) and resuspended in ChIP lysis buffer. The cell lysate was centrifuged at 10,000 × g for 10 min at 4°C. Anti-immunoglobulin G (IgG) (product code ab131368; Abcam) or anti-SND1 antibody (product code ab65078; Abcam) were incubated with Dynabeads protein G (cat. no. 10003D; Life Technologies; Thermo Fisher Scientific, Inc.) for 2 h at 4°C, followed by incubation with precleared chromatin overnight at 4°C. The precipitated RNA samples and inputs were subjected to RT-qPCR.

Zhao Y., Ren P., Yang Z., Wang L, & Hu C. (2022). Inhibition of SND1 overcomes chemoresistance in bladder cancer cells by promoting ferroptosis. Oncology Reports, 49(1), 16.

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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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Chromatin Immunoprecipitation for miR-193b Analysis

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Cells (3×10⁷) were fixed with 1.5% formaldehyde for 10 min at room temperature and quenched with glycine. After cell lysis with RIPA buffer (Beyotime Institute of Biotechnology), chromatin was fragmented into 100–500 bp fragments using an ultrasonic cell breaker (DH92-IIN; Lawson Scientific Ltd.) with a cycle of 1 sec on/1 sec off for 8 min (frequency, 20 kHz). Protein-DNA complexes were immunoprecipitated with MYC antibody (cat. no. ab32072; Abcam) or anti-IgG antibody (cat. no. ab171870; Abcam) conjugated with Dynabeads Protein G (cat. no. 10007D; Invitrogen; Thermo Fisher Scientific, Inc.) as described previously (22 (link)). After being eluted from the beads using ChIP Elution Buffer (cat. no. 714231S; Cell Signaling Technology, Inc.) for 30 min at 65°C, the antibody-protein-DNA complex was reverse cross-linked by incubation at 65°C with 200 mM NaCl. Six primers were designed according to the predicted binding sites on miR-193b (sequences are listed in Table I), and quantification of immunoprecipitated DNA was performed by RT-qPCR. Each sample was tested in triplicate, and the experiment was repeated three times.

Gao J., Ma S., Yang F., Chen X., Wang W., Zhang J., Li Y., Wang T, & Shan L. (2020). miR-193b exhibits mutual interaction with MYC, and suppresses growth and metastasis of osteosarcoma. Oncology Reports, 44(1), 139-155.

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