GIST cells were transfected with pcDNA or pcDNA-HOTAIR. For immunoprecipitation of endogenous RNP complex, the cells were lysed with nuclear isolation buffer (1.28 M sucrose, 40 mM Tris-HCl [pH7.5], 20 mM MgCl2, 4% Triton X-100) and resuspended in RIP buffer (150 mM KCl, 25 mM Tris-HCl [pH7.4], 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 1U/ul RNAase inhibitor, protease inhibitor). The shearing of chromatin was mechanically conducted using a Dounce homogenizer with 15-20 strokes. After centrifugation, SUZ12 antibody (2ug) was added to supernatant (600-800ug) and incubated for 2 h at 4°C with gentle rotation. After incubation, Magna Chip protein A magnetic beads (Merck Millipore, Darmstadt, Germany) was added and incubated for 2 hrs at 4°C with gentle rotation. The pellet was washed with RIP buffer and repeated for a total of three RIP washes. After washing, each sample was lysed with TRizol reagent or eluted in SDS, and then analyzed on Western blot.
Magna chip protein a magnetic beads
Manufactured by Merck Group
Sourced in Germany, Canada, United States
Magna ChIP Protein A magnetic beads are a product designed for chromatin immunoprecipitation (ChIP) assays. The beads are coated with Protein A, which binds to the Fc region of antibodies, allowing for the capture and isolation of protein-DNA complexes. The magnetic properties of the beads enable easy separation and washing steps during the ChIP process.
Automatically generated - may contain errors
Lab products found in correlation
Ipure kit, by Diagenode (4 mentions)
Anti trimethyl h3k4, by Abcam (1 mentions)
Anti h2bub1, by Cell Signaling Technology (1 mentions)
Bioruptor plus sonication device, by Diagenode (1 mentions)
Anti p21 sc 397, by Santa Cruz Biotechnology (1 mentions)
Anti p27 c 19, by Santa Cruz Biotechnology (1 mentions)
Anti e2f4 sc 866, by Santa Cruz Biotechnology (1 mentions)
Flag antibody, by Merck Group (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Xl 2000, by Qsonica (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Anti ar, by Merck Group (1 mentions)
Anti ar, by Santa Cruz Biotechnology (1 mentions)
Anti p21, by Merck Group (1 mentions)
Ab77231, by Abcam (1 mentions)
Ab23738, by Abcam (1 mentions)
Ab109388, by Abcam (1 mentions)
Anti pias1, by Santa Cruz Biotechnology (1 mentions)
Anti tubulin, by Santa Cruz Biotechnology (1 mentions)
14 protocols using magna chip protein a magnetic beads
1
Immunoprecipitation of HOTAIR-associated RNP complex
2
Chromatin Immunoprecipitation in Plants
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ChIP experiments were performed as previously described (Saleh et al., 2008 ) with some modifications. A 3 g sample of leaves was fixed with cross‐linking buffer and 1% formaldehyde using vacuum infiltration two times for 15 min each, and the cross‐linking reaction was quenched with 0.125 m glycine. The leaves were ground in a mortar and pestle in liquid nitrogen, resuspended in nuclei isolation buffer and then filtered through Miracloth. After centrifugation, the pellets (nuclei) were resuspended in cold nuclei lysis buffer and sonicated (Bioruptor® Plus sonication device, Diagenode, Belgium). After centrifugation, the supernatant was pre‐cleared with Magna ChIP™ Protein A Magnetic Beads (EMD Millipore corporation, Temecula, CA), and specific antibodies were added and incubated overnight at 4 °C. The specific antibodies used were as follows: anti‐H2Bub1 (Cell Signaling Technology, Danvers, MA) and anti‐trimethyl‐H3K4 (Abcam). The enriched DNA fragments were detected by RT‐qPCR and compared with the input samples, the amount of immunoprecipitated chromatin as normalized to the total amount of chromatin used in GhDREB P1 region in wild‐type plants was given as 1. GhUBI1 was used as a negative control.
3
ChIP Assay for Protein-DNA Interactions
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HCT116 cells were asynchronously grown till achieve an 80% confluence. ChIP assays were then performed as previously described (18 (link)). Briefly, cells were lysed and chromatin from cross-linked cells was sonicated. Chromatin was incubated with 5 μg of anti-p27, anti-PCAF or anti-PAX5 antibodies in RIPA buffer (50 mM Tris−HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mM Na3VO4, 0.5 μg/μl aprotinin, 10 μg/μl leupeptin) adding 20 μl of Magna ChIP Protein A magnetic beads (Millipore). Samples were incubated in rotation overnight at 4°C. Beads were washed with low salt buffer, high salt buffer, LiCl buffer and TE buffer. Subsequent elution and purification of the immunoprecipitated DNA-proteins complexes was performed using the IPure kit (Diagenode) according to manufacturer's protocol. Samples were analyzed by qPCR. The association of p27 and PCAF to their respective binding sites of the gene Grin3A, SPAG9, ROBO1 and UNC5D was performed by RT-PCR using the primers listed in Supplementary Table S1 .
4
Chromatin Immunoprecipitation Assay for p27, p21, and E2F4
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c17.2 cells were grown in an 80% confluence plate. Chromatin immunoprecipitation (ChIP) assays were performed as previously described [39 (link)]. Briefly, cells were lysed and chromatin from crosslinked cells was sonicated. Chromatin was incubated with 5 μg of anti-p27 (C-19, Santa Cruz), anti-p21 (sc-397, Santa Cruz) or anti-E2F4 (sc-866, Santa Cruz) in RIPA buffer (50 mM Tris - HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mM Na3VO4, 0.5 μg/μl aprotinin, 10 μg/μl leupeptin) adding 20 μl of Magna ChIP Protein A magnetic beads (Millipore). Samples were incubated in rotation overnight at 4°C. Samples incubated without antibodies were used as a control. Beads were washed with low-salt buffer, high-salt buffer, LiCl buffer and TE buffer. Subsequent elution and purification of the immunoprecipitated DNA-protein complexes was performed using the IPure kit (Diagenode) according to manufacturer’s protocol. Samples were analysed by qPCR. Primer sequences used for qPCR of SNCA promoter genomic regions are listed in Supplementary Table 1 .
5
ChIP Assay for Cell Cycle Regulators
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Cells were grown to confluence and synchronized in quiescence or at different times of G1 phase. ChIP assays were performed as previously described (25 (link)). Briefly, cells were lysed and chromatin from cross-linked cells was sonicated. Chromatin was incubated with 2.5–5 μg of antibodies against p27 (sc-528, Santa Cruz), p21 (sc-397), cyclin D1 (sc-246), cyclin D2 (sc-181), cyclin D3 (sc-182), Cdk4 (sc-260) or Cdk2 (sc-6248) in RIPA buffer, adding 20 μl of Magna ChIP Protein A magnetic beads (Millipore). Samples were incubated in rotation overnight at 4ºC. Beads were washed with low salt buffer, high salt buffer, LiCl buffer and TE buffer. Subsequent elution and purification of the immunoprecipitated DNA–proteins complexes was performed using the IPure kit (Diagenode) according to manufacturer's protocol. Samples were analyzed by quantitative PCR (qPCR). Primer sequences used for qPCR were listed in Supplementary Table S1.
6
Chromatin Immunoprecipitation of E2F1
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A549 cells were treated with formaldehyde and incubated for 10 min to generate DNA-protein crosslinks. Cell lysates were then sonicated to generate chromatin fragments of 200-300 bp and immunoprecipitated with E2F1-specific antibody (Cell Signaling Technology) or IgG as a control. Precipitated chromatin DNA was recovered and analyzed by qPCR and the amplified DNA was analyzed by agarose (2%) gel electrophoresis for visualization. The ChIP assays were performed using a Magna ChIP™ Protein A Magnetic Beads (Cat. 17-610, Millipore, USA), according to the manufacturer's instructions. The promotor primers of PTTG3P used for the analysis of E2F1 binding are listed in Supplemental Table S2 .
7
ChIP Analysis of α-Synuclein Binding
8
Immunoblotting and Immunoprecipitation of PIAS1, AR, and FOXA1
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VCaP cell samples were prepared as described (27 (link)) and analyzed by immunoblotting with anti-PIAS1 (Santa-Cruz Biotechnology, sc-8152), anti-AR (28 (link)), anti-p21 (Millipore, OP64) and anti-tubulin (Santa-Cruz Biotechnology, sc-5286) antibodies. The appropriate secondary antibody was from Invitrogen and chemiluminescence detection reagents from Pierce. PIAS1 was immunoprecipitated with anti-PIAS1 antibody (Abcam, ab109388) which was coupled to Magna ChIP™ Protein A Magnetic Beads (Millipore). Normal rabbit IgG (Santa-Cruz Biotechnology, sc-2027) was used as a control antibody and the immunoprecipitates were immunoblotted with anti-AR (Santa-Cruz Biotechnology, sc-7305) antibody. For FOXA1 and PIAS1 immunoprecipitation, anti-FOXA1 (Abcam, ab23738) antibody was used to immunoprecipitate FOXA1 and the immunoprecipitates were immunoblotted with anti-PIAS1 (Abcam, ab77231) antibody.
9
H3K27me3 Chromatin Immunoprecipitation
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Histones ChIP analysis was performed by using 5 μg of H3K27me3 antibody (07-449; Millipore Corp.) or rabbit IgG (Millipore Corp.) with magnetic beads (Magna ChIP protein A magnetic beads; 16661; Millipore). After washes, samples were eluted with the elution buffer (TE 1x, sodium dodecyl sulfate 0.5%), treated with RNAse A and with proteinase K. The extracted DNA was used in the qPCR analyses (primers are listed in Table 3 ). Data were expressed as (2(−ΔCt)) x100 (% Input).
10
Histone Modification Profiling by ChIP
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ChIP experiments for histone modifications were performed using Magna ChIP Protein A Magnetic Beads (Millipore) according to the manufacturer's instructions and using chromatin amounts corresponding to 50 µg of DNA. The antibodies used were: anti-Acetyl-Histone H3 (α-AcH3, 06-599; Millipore), anti-trimethyl-Histone H3 (Lys4) (α-H3K4me3, 07-473; Millipore), Anti-Histone H3 (acetyl K9, phospho S10) (α-pAcH3, ab12181; Abcam) and Normal Rabbit IgG (12–370; Millipore). Pulled-down DNA was analyzed in triplicate by qPCR and the values were normalized respect to GAPDH promoter (used as control region). Each experiment was repeated at least twice and a representative result is shown. The negative control values (not shown) were lower than the 10% of the immunoprecipitated samples.
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