MDA-iPRA cells were cultured for 36–48 h in DMEM/charcoal-stripped fetal bovine serum followed by 1 h treatment with P4 (10 nM) and/or UPA (1 μM). ChIP assays were performed as previously described [28 ] (#HighCellChIP kit, Diagenode, Seraing, Belgium) with 5 μg of the appropriate ChIP grade antibodies: Human anti-PR (Anti-PR, sc-7208, Santa Cruz Biotechnology, CA); Rabbit anti-SRC1 antibody (M-341, sc-8995,Santa Cruz Biotechnology), rabbit anti-Polymerase II antibody (H-224, sc-9001, Santa Cruz Biotechnology) and control unrelated antibody from the #HighCellChIP kit (Diagenode).
Highcell chip kit
Manufactured by Diagenode
Sourced in Belgium, United States
The HighCell ChIP kit is a laboratory equipment designed for chromatin immunoprecipitation (ChIP) experiments. It is used to isolate and analyze DNA-protein interactions within a cell. The kit provides the necessary reagents and protocols to perform ChIP from a small number of cells.
Automatically generated - may contain errors
Lab products found in correlation
Bioruptor, by Diagenode (8 mentions)
Formaldehyde, by Merck Group (3 mentions)
Ipure kit, by Diagenode (2 mentions)
Bioruptor plus, by Diagenode (1 mentions)
Anti parp1 monoclonal antibody, by Cell Signaling Technology (1 mentions)
Lc480, by Roche (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Anti creb, by Abcam (1 mentions)
Sybr green reagent, by Thermo Fisher Scientific (1 mentions)
Sonicator, by Diagenode (1 mentions)
Anti brd4, by Fortis Life Sciences (1 mentions)
Anti gr antibody m20 sc 23476, by Santa Cruz Biotechnology (1 mentions)
Protein a coated magnetic beads, by Diagenode (1 mentions)
Normal igg, by Merck Group (1 mentions)
Ab 105 c, by R&D Systems (1 mentions)
Bioruptor pico sonicator, by Diagenode (1 mentions)
H 224 sc 9001, by Santa Cruz Biotechnology (1 mentions)
Anti evi1, by Cell Signaling Technology (1 mentions)
Q700 sonicator, by Qsonica (1 mentions)
Ab6002, by Abcam (1 mentions)
24 protocols using highcell chip kit
1
Progesterone Receptor Chromatin Immunoprecipitation
2
ChIP Assay for Transcription Factor Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assay was performed according to the protocol specified by the manufacturer (HighCell# ChIP kit, Diagenode Inc.), In brief, treated cells were collected by trypsinisation, and DNA-protein interactions were cross-linked with 1% formaldehyde. Chromatin shearing was performed by sonication at 4°C with a Bioruptor® Plus equipped with a Minichiller (Diagenode Inc.). Lysates were centrifuged, and aliquots (1%) of the supernatants were collected for input control, while the remaining supernatants were incubated overnight (at 4°C) with magnetic beads coated with ChIP grade antibodies against p65, AhR or Arnt. DNA was isolated from the collected precipitates after reversal of the cross-linking by incubation for 15 min at 55°C. Real-time PCR were performed using primer sets for human CXCL8 and CCL5 promoter regions containing NF-κB response elements [62 (link)],[63 (link)]. The sequences of primers used for ChIP assay were as follows: human CXCL8 sense 5’-AGTGTGATGACTCAGGTTTGCCCT-3’ and anti-sense 5’-AAGCTTGTGTGCTCTGCTGTCTCT-3’; human CCL5 promoter sense 5’-GGGAAGAAGATTGCCTAAAC-3’ and antisense 5’-TGTGGAAATCAAAGGGACAG-3’.
3
ChIP-qPCR Protocol for Epigenetic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected and subjected to ChIP according to the manufacturer's instructions (HighCell#ChIP kit, Diagenode, NJ) using anti‐PARP1 monoclonal antibody (Cell Signaling), anti‐mouse CHD1L polyclonal antibody (LTK BioLaboratories), anti‐histone H3 monoclonal antibody (ChIP Formulated; Cell Signaling), and anti‐di‐methyl‐histone H3 (Lys4) monoclonal antibody (Cell Signaling). DNA eluted from precipitated complexes was analyzed by qPCR. qPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, CA) according to the manufacturer's instructions. Signals were detected with LC480 (Roche, Mannheim, Germany). Primer sequences are listed in Supporting Information Materials and Methods.
4
ChIP-qPCR Analysis of SATB1 and CREB Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MGE cells were treated with activin A (100 ng/ml) for 60 min, collected by trypsinization, and washed twice in ice-cold PBS. Cells of 3 wells from 12-well plates were pooled together (∼450,000 cells in total). DNA–protein complexes were cross-linked by incubating the cells with 37% formaldehyde (final concentration 1%) for 8 min. DNA was then purified and immunoprecipitated using a HighCell ChIP Kit (Diagenode) according to the manufacturer’s protocol. DNA was sheared by sonication using a Sonicator (Diagenode): 10 cycles (30 s on, 30 s off) at high power setting. DNA was immunoprecipitated using an anti-SATB1 antibody (Santa Cruz, X-5889, 6.3 µg/sample), or anti-CREB (Abcam, 31387, 6.3 µg/sample) or mouse-IgG (provided with the kit). Immunoprecipitates were amplified and analyzed by real-time quantitative PCR (qPCR) using SybrGreen reagents (Applied Biosystems). The following qPCR primers were used: SST-promoter-R1: Fw: 5′-CCTGTAGGGATCATCTCGTCC-3′, Rv: 5′-GGCCAGAGTTCTGACTGCTTT3′; SST-promoter-R2: Fw 5′-ACTCTGGCCTGAACAGTAAACAT3′, Rv: 5′-TCAGCTCTGCCTGATCTCCTA3′; and IL2a-promoter: Fw 5′-GGGGGTGGGGATACAAAGTAA3′, Rv: 5′-TCTTGCTCTTGTCCACCACAATA3′.
5
Chromatin Immunoprecipitation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ChIP assays were performed using a HighCell ChIP kit with IPure 2.0 kit elution (Diagenode) according to the manufacturer's protocol. The antibodies used for ChIP assays are AR_PG-21 (Millipore Cat. # 06-680); TBP (Abcam Cat. #ab63766), and Normal Rabbit IgG Control (Diagenode). LNCaP cells were grown in charcoal-stripped serum containing media for 48 h followed by 12 h of vehicle or 1 nM R1881 and dox treatment (0.1 μM for ‘low,’ 0.4 μM for ‘medium,’ and 0.7 μM for ‘high’) for all ChIP-qPCR and ChIP-seq conditions. ChIP-seq sample preparation for sequencing was performed according to the manufacturer's instructions (Illumina). Further experimental details as well as information on ChIP-seq enrichment analysis and overlaps of AR binding are in Asangani et al. (32 (link)). Transcriptional motifs were identified using MEME or DREME (33 (link)).
6
Chromatin Immunoprecipitation in MCF7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HighCell# ChIP kit (Diagenode) was utilized to perform ChIP assays via the manufacturer's protocol. Briefly, MCF7 cells were grown in charcoal-stripped serum media (described above) for 72 h and then stimulated 10 nM estradiol for 12 h. Cells were then crosslinked using 1% formaldehyde for 10 min, and crosslinking was quenched for 5 min at room temperature using a 1/10 volume of 1.25 M glycine. Cells were then lysed and sonicated (Bioruptor, Diagenode), yielding an average chromatin fragment size of 300 bp. An equivalent amount of chromatin equivalent to 5 × 106 cells was used for the ChIP for all antibodies. DNA bound to immunoprecipitated product was isolated (IPure Kit, Diagenode) via overnight incubation with antibody at 4 °C. Samples were then washed, and crosslinked reversed.
7
ChIP-seq Protocol with HighCell# Kit
8
ChIP Assay of Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP was performed following the HighCell# ChIP Kit protocol from Diagenode (C01010063 (kch-mahigh-G48)). 6 µg of the anti-acetyl-Histone H4 (Lys16) (#07–329, EMD Millipore), anti-KAT8/MYST1/MOF (ab200660, Abcam), or anti-H3 C-terminal (Active Motif, 39163) were used in each immunoprecipitation. Predesigned primers focusing on the 1 kb genomic regions in gene promoters for mouse IL-1β (NM_008361.3 (+) 01 kb) and IL6 (NM_031168.1 (+) 01 kb) and Mmp14 (NM_008608.2 (+) 01 kb) and Ccl22 (NM_009137.1 (+) 01 kb) and Chil3 (NM_009892.1 (+) 01 kb) used for ChIP were purchased from Qiagen (EpiTect ChIP qPCR Primer Assay for mouse). They were used at 5 µM/well and they target 1-kb region downstream of the transcription start sites (TSS).
9
Chromatin Immunoprecipitation Protocol for BRD4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidneys were fixed in 1% formaldehyde (Sigma-Aldrich), followed by quenching with 0.125M glycine. DNA fragments 500–1000 bp long were generated on a BioRuptor (Diagenode; Denville, NJ, USA), and ChIP assays were performed using the High Cell ChIP kit (Diagenode) following the manufacturer’s instructions. The antibodies used were anti-BRD4 (Bethyl Laboratories; Montgomery, TX, USA) and normal IgG as negative control (Millipore). Immunoprecipitated DNA was analyzed by quantitative RT-PCR using the Col4a3 primers: Forward: 5′-CCACTTCTCCCCTCCCTTAG-3′//Reverse 5′-CCCGGTGTTTTCTGTGTTCT -3′. Chromatin obtained before immunoprecipitation was used as the input control. Relative enrichment was calculated as the percentage of input DNA for each sample using the formula % input = 2 exp [(Ct unbound) − log2 (unbound dilution factor) − Ct bound)] × 100 and normalized to normal rabbit IgG antibody (considered as 1).
10
Immunoprecipitation of Glucocorticoid Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!