17-295 Sigma-AldrichChromatin Immunoprecipitation (ChIP) Assay Kit
Contains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using inexpensive protein A agarose beads.
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Overview
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HS Code | 3822 19 90 |
Presentation | Contains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions. Supplied buffers are sufficient to generate chromatin from up to five 15 cm plates of cultured cells, each plate providing up to 10 chromatin preparations (varies with cell and assay type). |
Quality Level | MQ100 |
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Application | Contains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using inexpensive protein A agarose beads. |
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Material Size | 1 kit |
Material Package | Kit capacity: 22 Assays |
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Catalogue Number | GTIN |
17-295 | 08436037124679 |
Documentation
Chromatin Immunoprecipitation (ChIP) Assay Kit SDS
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Chromatin Immunoprecipitation (ChIP) Assay Kit Certificates of Analysis
References
Reference overview | Application | Species | Pub Med ID |
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HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic neurogenesis and in schizophrenia-like animals. Večeřa, J; Bártová, E; Krejčí, J; Legartová, S; Komůrková, D; Rudá-Kučerová, J; Štark, T; Dražanová, E; Kašpárek, T; Šulcová, A; Dekker, FJ; Szymanski, W; Seiser, C; Weitzer, G; Mechoulam, R; Micale, V; Kozubek, S J Cell Physiol 233 530-548 2018 Show Abstract | 28300292
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Differential in Vitro Biological Action, Coregulator Interactions, and Molecular Dynamic Analysis of Bisphenol A (BPA), BPAF, and BPS Ligand-ERα Complexes. Li, Y; Perera, L; Coons, LA; Burns, KA; Tyler Ramsey, J; Pelch, KE; Houtman, R; van Beuningen, R; Teng, CT; Korach, KS Environ Health Perspect 126 017012 2018 Show Abstract | 29389661
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RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis. Cui, XB; Luan, JN; Dong, K; Chen, S; Wang, Y; Watford, WT; Chen, SY Arterioscler Thromb Vasc Biol 38 e36-e47 2018 Show Abstract | 29449334
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Tumor-secreted Pros1 inhibits macrophage M1 polarization to reduce antitumor immune response. Ubil, E; Caskey, L; Holtzhausen, A; Hunter, D; Story, C; Earp, HS J Clin Invest 128 2356-2369 2018 Show Abstract | 29708510
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Nuclear IGF-1R interacts with regulatory regions of chromatin to promote RNA polymerase II recruitment and gene expression associated with advanced tumor stage. Aleksic, T; Gray, NE; Wu, X; Rieunier, G; Osher, E; Mills, J; Verrill, C; Bryant, RJ; Han, C; Hutchinson, K; Lambert, A; Kumar, R; Hamdy, FC; Weyer-Czernilofsky, U; Sanderson, M; Bogenrieder, T; Taylor, S; Macaulay, VM Cancer Res 0 2018 Show Abstract | 29735545
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miR-4725-3p targeting Stim1 signaling is involved in xanthohumol inhibition of glioma cell invasion. Ho, KH; Chang, CK; Chen, PH; Wang, YJ; Chang, WC; Chen, KC J Neurochem 0 2018 Show Abstract | 29747239
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Transcription factor NF-kappa B represses ANT1 transcription and leads to mitochondrial dysfunctions. Zhang, C; Jiang, H; Wang, P; Liu, H; Sun, X Sci Rep 7 44708 2017 Show Abstract | 28317877
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The chromatin remodelling factor ATRX suppresses R-loops in transcribed telomeric repeats. Nguyen, DT; Voon, HPJ; Xella, B; Scott, C; Clynes, D; Babbs, C; Ayyub, H; Kerry, J; Sharpe, JA; Sloane-Stanley, JA; Butler, S; Fisher, CA; Gray, NE; Jenuwein, T; Higgs, DR; Gibbons, RJ EMBO Rep 18 914-928 2017 Show Abstract | 28487353
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Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo. Hanssen, LLP; Kassouf, MT; Oudelaar, AM; Biggs, D; Preece, C; Downes, DJ; Gosden, M; Sharpe, JA; Sloane-Stanley, JA; Hughes, JR; Davies, B; Higgs, DR Nat Cell Biol 19 952-961 2017 Show Abstract | 28737770
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DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease. Jeziorska, DM; Murray, RJS; De Gobbi, M; Gaentzsch, R; Garrick, D; Ayyub, H; Chen, T; Li, E; Telenius, J; Lynch, M; Graham, B; Smith, AJH; Lund, JN; Hughes, JR; Higgs, DR; Tufarelli, C Proc Natl Acad Sci U S A 114 E7526-E7535 2017 Show Abstract | 28827334
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Brochure
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An Introduction to Antibodies and Their Applications |
Shaping Epigenetics Discovery - Epigenetics Product Selection Brochure |
Data Sheet
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Reprogramming Cell Fate and Function Novel Strategies for iPSC Generation, Characterization, and Differentiation |
FAQ
Question | Answer |
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Can I alter the time and temperature of cross-link reversal? | Reversal of cross-links for shorter than four hours is not recommended. However, it is possible to leave the samples at 65° C overnight for cross-link reversal provided steps are taken to insure that the samples do not dry out. |
At times, when I try to pellet my beads, they don't form a tight pellet and it is difficult to remove the supernatant. This happens even after I spin longer and/or faster. What can I do the remedy this? | High-speed centrifugation is not necessary to pellet agarose beads. Often high-speed centrifugation will result in breaking agarose into smaller beads called 'fines'. If you are having trouble removing the supernatant from your pellet, you could use a small gel loading tip or a needle and syringe. This will allow you to remove the supernatant without disrupting the pellet. |
Do I have to phenol/chloroform my samples prior to PCR? | Not necessarily, but it is recommended. |
How should I resuspend my pellet prior to PCR? | You should resuspend your pellet in water and not TE as the EDTA found in the TE may interfere with PCR. |
Is there ever a time when I do not need to cross-link Histones? | In native ChIP, Histone H3 and Histone H4 do not need to be crosslinked as they are very tightly associated. Histone H2A and Histone H2B are not as tightly associated, but will still work in native ChIP. |
What were your conditions for PCR? | Please see the manual for The EZ ChIP Kit (Catalog #17-371) for more information. |
If I wanted to quantitate my immunoprecipitated DNA, how would I do so? | DNA purified from ChIP experiments can be quantitated by PCR, providing the amplifying oligos meet specific criteria. Oligos should be 24 mers, with a GC content of 50% (+/- 4) and a Tm of 60.0C (+/- 2.0). You must be certain that the PCR reactions are within the linear range of amplification. Generally it takes time to achieve this. Too much input DNA will affect your results, so set up several tubes for each experiment to optimize the input DNA. Generally, this is about 1/25th to 1/100th for yeast, approximately 1/10 for mammalian cells, but depends on the amount of antibody and input chromatin. Also, do not use more than 20 cycles, making sure that dNTP's always remain in excess. Also, include each reaction a control primer (to compare your experimental band against-make sure the sizes are sufficiently different to allow proper separation-75 base pairs is usually OK) set to a region of the genome that should not change throughout your experimental conditions. Also PCR from purified input DNA (no ChIP) and include no antibody control PCR's as well. PCR products should be no more than 500 base pairs and should span the area of interest (where you think you will see changes in acetylation or methylation of histones). All PCR products should be run on 7-8% acrylamide gels and stained with SYBR Green 1 (Molecular Probes) at a dilution of 1:10,000 (in 1X Tris-borate-EDTA buffer, pH 7.5) for 30 minutes-no destaining is required. Quantitation is carried out subsequent to scanning of the gel on a Molecular Dynamics Storm 840 or 860 in Blue fluorescence mode with PMT voltage at 900 with ImageQuant software. This has distinct advantages over ethidium bromide staining. SYBR Green is much more sensitive, and illumination of ethidium stained gels can vary across the gel based on the quality of UV bulbs in your in your light box. For further info, see Strahl-Bolsinger et al. (1997) Genes Dev. 11: 83-93. A radioactive quantitation m |
I am not getting amplification with input DNA. What did I do wrong? | Your input DNA sample should be taken just prior to adding the antibody. It is considered the starting material. If you are not seeing amplification with your input DNA, either you have not successfully reversed the cross links or the PCR is not working for reasons other than the kit. |
How would you recommend eluting Antibody-protein-DNA complexes from agarose (or sepharose) in order to perform a Re-ChIP experiment? | The complex is removed with the elution buffer that you find in the ChIP assay kit. For a re-CHIP, it might make sense to add protease inhibitors to the IP wash buffers and the elution buffer and the second set of dilution buffers. Make sure everything stays cold so that the proteins aren't degraded during the collection of the first complex or during the second IP. |
Do you have any tips for sonication? | Keep cells on ice throughout the procedure - even during sonication. Be sure that you don't sonicate for to long (more than 30 seconds could cause sample overheating and denaturation). |