ChIP assays were performed as described previously (Bao et al., 2017 (link)). Briefly, BMDMs (∼2 × 107 cells) were cross-linked with 1% formaldehyde for 10 min at room temperature. Cross-link was quenched by 125 mM glycine, and cells were sonicated with a Diagenode Bioruptor 300 to reach the desired genomic fragment length (∼300–700 bp). Immunoprecipitation was performed overnight at 4°C by mixing cell lysates, antibodies of interest, and Protein A Magnetic Dynabeads (10001D; Thermo Fisher Scientific). Protein–DNA complexes were reverse cross-linked at 65°C overnight with the addition of proteinase K (0.2 mg/ml). DNAs were further purified with a Qiagen quick spin column, and real-time PCR was performed as described above. Primer sequences used for the ChIP-PCR were as follows: Naip1 (forward, 5′-ATAGCCTGGCCCAATTCTTT-3′; reverse, 5′-GGCTTGGCAGCTTTGATTAG-3′), Naip2 (forward, 5′-CAGCAAGGGGGCAGAGAAAAT-3′; reverse, 5′-ACAGGCAGCTCATGGTTTGAG-3′), and Naip5/Naip6 (forward, 5′-TATAGCCTGGTGCCACTTCC-3′; reverse, 5′-AACCCTGACAAAAGCAGTTCA-3′).
Protein a magnetic dynabeads
Manufactured by Thermo Fisher Scientific
Protein A Magnetic Dynabeads are superparamagnetic beads coated with recombinant Protein A. They are designed for the rapid and efficient purification of antibodies from cell culture supernatants, ascites fluid, and other sample types.
Automatically generated - may contain errors
Lab products found in correlation
Quick spin column, by Qiagen (2 mentions)
Bioruptor 300, by Diagenode (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Rabbit anti m6a, by Synaptic Systems (1 mentions)
Proteinase k, by New England Biolabs (1 mentions)
Suz12, by Abcam (1 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Ribolock, by Thermo Fisher Scientific (1 mentions)
Rabbit anti caprin 1, by Proteintech (1 mentions)
Rabbit anti igg, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
H3k27ac antibody, by Active Motif (1 mentions)
Sonic dismembrator, by Thermo Fisher Scientific (1 mentions)
8 protocols using protein a magnetic dynabeads
1
ChIP-PCR Assay for NAIP Genes
2
m6A RNA Immunoprecipitation Protocol
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DNA was denatured by incubating at 95°C for 10 min and then immediately placed on ice for 5 min. 200 μl of cold water and 500 μl of cold 5× DIP buffer (50 mM NaPO4, pH 7.0, 700 mM NaCl, 0.25% Triton X-100) were added to bring the volume up to 1 ml. 50 μl was taken aside as input. 25 μl of 20 mg/ml BSA and 1.8 μg of antibody (Synaptic Systems rabbit anti-m6A, Cat 202–003) were added to the rest of the sample and rotated overnight at 4°C. 50 μl of Protein A magnetic Dynabeads (Thermo Fisher, Cat # 10,002D) were added to the sample and rotated at 4°C for 1 hr. Magnetic beads were immobilized using a magnetic rack and washed by resuspension and rotation for 5 min at 4°C in 1 ml of various cold buffers in the following order: 2× with 1× DIP buffer + 0.05% Tween-20, 1× with 1× DIP buffer. For elution, beads were resuspended in 190 μl of DIP digestion buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 0.5% SDS) + 10 μl of 10 mg/ml Proteinase K (New England Biolabs, Cat # P8107S). DIP digestion buffer was added to input samples up to 200 μl. Both the input and IP samples were incubated at 50°C for 2 hr and then cleaned up using the Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany, Cat # 28104) and eluted in 35 μl of water.
3
ChIP Assays of Immortalized BMDMs
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ChIP assays were performed as described previously.18 Briefly, immortalized BMDMs (∼2 × 107 cells) were cross-linked with 1% formaldehyde for 10 minutes at room temperature. The cross-link was quenched by 125 mmol/L glycine and cells were sonicated with the Diagenode Bioruptor 300 (Diagenode) to reach the desired genomic fragment length (∼300–700 bp). Immunoprecipitation was performed overnight at 4°C by mixing cell lysates, antibodies of interest, and Protein A Magnetic Dynabeads (10001D; Thermo Fisher Scientific). Protein–DNA complexes were reverse cross-linked at 65°C overnight with the addition of proteinase K (0.2 mg/mL). DNAs were purified further with the Qiagen quick spin column and quantitative PCR was performed as described earlier.
4
Chromatin Immunoprecipitation (ChIP) Protocol
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Cells (3×106/ChIP) were cross-linked with 1% formaldehyde for 10 min. The cross-linked chromatin was sonicated (30sec ON/OFF for 10 min in a Bioruptor®Pico (Diagenode)) or enzymatically digested (EZ-ZymeTM Chromatin Prep kit (Cat#17-375, Millipore) to generate <500 bp DNA fragments. Chromatin supernatant (1%) was collected as the input before adding the following immunoprecipitating antibodies: H3K4me3 (Diagenode C15410003), H3K27me3 (Diagenode C15410069 or Abcam ab6002), H3K27ac (Diagenode C15410174), H3K4me1 (Diagenode C15410037) and SUZ-12 (Abcam ab12073). Magnetic Dynabeads Protein A (Invitrogen) or protein G Agarose (EZ-ChIPTM Chromatin Immunoprecipitation kit (Cat#17-371, Millipore)) were used to bind the antibody/antigen/DNA complex. ChIP samples were washed, crosslinks were reversed and ChIP DNA was isolated as the template for real-time qPCR using either SYBR Green (BioRad) or custom designed TaqMan assays (Tables S5 and S6 ).
5
Magnetic Bead-Based SARS-CoV-2 Antigen Capture
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Magnetic Dynabeads Protein A (Invitrogen) were washed with PBS using a magnetic tube rack. Beads were incubated with COVA-18 at 0.7 μg antibody per 1 μL beads for 2 h at room temperature in an overhead rotator. Excess antibodies were removed by washing the beads with PBS. One μg of SARS-CoV SARS-CoV-2 mosaic I53-50 NP was added for every μg of antibody and the mixture was incubated at room temperature in an overhead rotator overnight. Beads were washed with PBS to remove unbound particles and protein was eluted by boiling for 10 min in PBS.
6
Caprin-1 Immunoprecipitation in HeLa Cells
7
ChIP-seq Analysis of H3K27ac in Prostate Cancer
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ChIP on prostate cancer biopsy and prostatectomy tissues were performed as previously described [61 (link)]. Nuclear lysates of each tissue specimen were incubated with 5 μg of H3K27ac antibody (Active Motif, 39133) pre-bound to 50 μL magnetic protein A Dynabeads (Thermo Fisher Scientific, 10008D). Immunoprecipitated DNA was processed for library preparation (Part# 0801-0303, KAPA biosystems kit), and samples were sequenced using an Illumina HiSeq 2500 system (65 bp, single-end). Sequences were aligned to the human reference genome hg19, duplicate reads were removed, and reads were filtered based on MAPQ quality (≥ 20).
8
ChIP-qPCR for HIF-2α Targets
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