Mice were injected 1 μg recombinant IL-22 (Peprotech) or PBS i.p. 1h prior to epithelial cell isolation. Epithelial cells were isolated as described above. Cells were resuspended in PBS and fixed with 0.75% paraformaldehyde. Fixation was stopped with glycine and cells were washed and lysed in 50mM Tris, 2.5mM EDTA, 0.1% NP-40 and 10 % Glycerol. Nuclei were lysed in 50mM Tris, 5mM EDTA and 0.25% SDS and subsequently sonicated in a Bioruptor Plus (Diagenode). Lysates were cleared by centrifugation and stored at -80°C. Protein-A-coated magnetic beads (Diagenode) were blocked with yeast tRNA (Life Technologies) and BSA (New England Biolab). Nuclear lysates were split in 10% input and 90% immunoprecipitation solutions. The latter was incubated with α-STAT3 (C-20; Santa Cruz Biotechnology) overnight rotating at 4°C. Protein-A coded magnetic beads were added and incubated for another 30min rotating at 4°C. Beads were purified and washed repeatedly. Bound DNA was eluted in 10mM TRIS, 1 mM EDTA and 2% SDS at 37°C for 15min. De-crosslinking was performed overnight at 65°C. RNA was removed by 1h incubation with RNAse A (Peqlab) at 37°C, followed by 1h incubation with Proteinase K (Peqlab) at 42°C. DNA was purified using PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. All antibodies used are listed in Supplementary Information Table 2 .
Protein a coated magnetic beads
Manufactured by Diagenode
Protein-A-coated magnetic beads are a type of lab equipment used for the purification and isolation of antibodies from biological samples. The beads are coated with Protein A, a bacterial protein that binds to the Fc region of immunoglobulins, allowing for the selective capture and separation of antibodies from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant il 22, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by New England Biolabs (2 mentions)
Yeast trna, by Thermo Fisher Scientific (2 mentions)
α stat3 c 20, by Santa Cruz Biotechnology (2 mentions)
Rnase a, by Avantor (2 mentions)
Proteinase k, by Avantor (2 mentions)
Pcr purification kit, by Qiagen (2 mentions)
Bioruptor plus, by Diagenode (2 mentions)
Bioruptor next gen, by Diagenode (1 mentions)
Sx 8g ip star robot, by Diagenode (1 mentions)
Highcell chip kit, by Diagenode (1 mentions)
Anti gr antibody m20 sc 23476, by Santa Cruz Biotechnology (1 mentions)
Sonifier 250, by Emerson (1 mentions)
Ideal chip qpcr kit, by Diagenode (1 mentions)
5 protocols using protein a coated magnetic beads
1
STAT3 Chromatin Immunoprecipitation in Epithelial Cells
2
Methylation-Specific DNA Enrichment and Analysis
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Methyl-CpG immunoprecipitation (MCIp) was performed as described previously [24 (link)]. In brief, a total of 2.5 μg DNA was sonicated with the Bioruptor NextGen (Diagenode, Liege, Belgium) to fragments of 100 to 600 bp as monitored on a 1.5% agarose gel. MCIp enrichment of highly methylated DNA was performed, as described, with minor modifications using SX-8G IP-Star robot (Diagenode). Sonicated DNA was enriched with 90 μg purified methyl-CpG-binding domain-Fc protein coupled to 60 μl protein A-coated magnetic beads (Diagenode). DNA was eluted by incubation with increasing NaCl concentrations (fraction A, 300 mM; B, 400 mM; C, 500 mM; D, 550 mM; E, 1,000 mM). Desalted eluates were controlled for enrichment of methylated DNA by real-time PCR analyzing the imprinted gene Mest. The non-methylated allele elutes at low-salt while the methylated allele elutes at high-salt concentration.
3
Immunoprecipitation of Glucocorticoid Receptor
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Immunoprecipitations were performed using 30 μl protein A-coated magnetic beads (Diagenode). Beads were washed 3 times with C1 buffer of the High Cell ChIP kit (Diagenode) and incubated for 4 h with anti-GR antibody H300 (Santa Cruz) at 4 °C with protease inhibitors and 0.2% BSA. Then, 350 μg of proteins from BZ cell lysates were incubated overnight with antibody-coated beads. The following day, beads were washed 3 times with buffer C1 and once with buffer W1 (High Cell ChIP kit), and immunoprecipitated proteins were eluted from the beads with 20 μl of Laemmli buffer at 95 °C for 5 min and loaded on a 10% SDS-PAGE gel for Western blotting. Membranes were hybridized with anti-GR antibody M20 (sc-23476, Santa Cruz, [54 (link)].
4
ChIP-qPCR Assay for Protein-DNA Interactions
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ChIP assays were performed using 1 million cells/IP. Cells were crosslinked in medium containing 1% formaldehyde at room temperature for 10 min with rotation. Formaldehyde was quenched by the addition of glycine (final concentration of 125 mM) for 5 min. Cell lysis, nuclei isolation, and immunoprecipitation (IP) were performed using the iDeal ChIP-qPCR kit following the manufacturer’ s recommendations (Diagenode). Chromatin fractions were sheared on ice for 10 × 10 s using a microtip (Branson Sonifier 250) to yield a DNA fragment size <1000 bp. Chromatin fragment size was monitored by agarose gel electrophoresis after DNA purification. After dilution of chromatin in ChIP reaction mix complemented with protease inhibitors cocktail, samples were incubated overnight at 4°C with the indicated relevant or control antibodies bound to 30 μl Protein A-coated magnetic beads (Diagenode). Beads were captured using a magnetic rack and sequentially washed before elution and DNA purification. Relative quantitation of target sequences in the input and the IP chromatin was performed by qPCR. The fold enrichment of a protein associated to a specific sequence was calculated with respect to the input DNA (1% of the ChIP fraction) and was compared with a ChIP signal obtained using a control non-relevant IgG.
5
STAT3 Chromatin Immunoprecipitation in Epithelial Cells
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