Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit

The use of different types of salts improves the effective removal of non-specific chromatin interactions with the agarose beads. Lithium is also soluble in buffers containing high amounts of SDS.

There are several points at which the procedure may be put on hold. A) After pelleting cells post formaldehyde and washes, you can freeze the cell pellet at -70° C. B) After making the lysate you can freeze at -70° C. C) After sonication, you can store the chromatin by aliquoting and storing at -70° C. D) After you reverse the cross-links prior to DNA purification you may store at -20° C.

As a gradual increase in stringency occurs, chromatin will be prevented from interacting with the beads. Unlike DNA hybridization, where lower salt equates with higher stringency, in the Chromatin IP, higher salt leads to higher stringency washing.

You can, please consult this protocol for additional information (This protocol is based upon protocols from Mark Biggin, Dave Allis and Richard Treisman) This protocol has successfully been used on livers, spleens, colons and whole mouse embryos. However, you may have to optimize conditions for your specific tissue type: Day 1: 1. Chop tissue into small pieces with a razor blade or scalpel. Transfer tissue into a tube with a screw cap lid and add a small (5-10mls) amount of tissue culture media. We have not noticed a difference with different types of media. 1X PBS should also work. Add formaldehyde to a final concentration of 1% and rotate tube at room temperature for 15 minutes. We generally use 0.03g of tissue per antibody. The exact amount of tissue that you will need depends upon how abundant your protein is, how strongly your antibody binds and how efficient your cross linking is. 2. Stop the cross linking reaction by adding glycine to a final concentration of 0.125 M. Continue to rotate at room temp for 5 minutes. 3. Centrifuge samples at low speed, decant media and wash once with cold 1X PBS. Centrifuge. Add a small (1-2mls per sample) amount of cold 1X PBS and disaggregate tissues. For this step, we use a Medimachine from Becton Dickinson. However, a dounce homogenizer also works. Centrifuge at low speed to pellet cells and decant supernatant. 4. Resuspend cell pellet in cell lysis buffer plus the protease inhibitors PMSF (10 ul per ml), aprotinin (1 ul per ml) and leupeptin (1 ul per ml). The final volume of cell lysis buffer should be sufficient so that there are no clumps of cells. Incubate on ice for 10-15 minutes. Cells can also be dounced on ice with a B dounce several times to aid in nuclei release. 5. Microfuge at 5,000 rpm for 5 minutes at 40 C to pellet the nuclei. 6. Resuspend nuclei in nuclei lysis buffer plus the same protease inhibitors as the cell lysis buffer. Incubate on ice for 10-20 minutes. 7. Add approximately 0.1 g of glass beads (Sigma G-1277) to each sample. Sonicate chromatin to an average length of about 600 bp while keeping samples on ice (the time and number of pulses will vary depending on sonicator, cell type and extent of cross linking). Microfuge at 14,000 rpm for 10 minutes at 40 C. At this point, chromatin can be snap frozen in liquid nitrogen and stored at -700 C for up to several months. 8. Carefully remove the supernatant and transfer to a new tube. Preclear chromatin by adding blocked Staph A cells. Use 10-15 uls of preblocked Staph A cells for every 0.3 g of tissue that you started with. 9. Incubate on a rotating platform at 40 C for 15 minutes, no longer. Microfuge at 14,000 rpm for 5 minutes. 10. Transfer supernatant to a clean tube and divide equally among your samples. Be sure to include a "no antibody" or pre-immune sample. We also include”mock” samples which contains 1X dialysis buffer instead of chromatin (no antibody and mock are critical to control for nonspecific interactions and DNA contamination of IP and wash solutions....the final output of this experiment is analyzed by PCR). Adjust the final volume of each sample with IP dilution buffer plus protease inhibitors if necessary. Samples volumes should be between 200 and 500 uls. Add 1 ug of antibody to each sample. 11. Incubate on the rotating platform at 40 C for at least 3 hours. Overnight is fine. If you are using monoclonal antibodies, add 1 ug of an appropriate secondary antibody and incubate for an additional 1 hour. Day 2: 12. Add 10 uls of blocked Staph A cells to each sample. Incubate on the rotating platform at room temp for 15 minutes, no longer. 13. Microfuge samples. Save the supernatant from the "no antibody" sample as "total input chromatin". 14. Wash pellets twice with 1.4 mls of 1X dialysis buffer (**if you are using a monoclonal antibody, omit the sarkosyl**) and four times with 1.4 mls of IP buffer (**pH 8.0 for monoclonal antibodies**). For each wash, dissolve the pellet in 200 uls of buffer and use an additional 200 uls of buffer to wash the pipette tip. Add an additional 1 ml of buffer. For each wash, incubate samples on a rotating platform for 3 minutes then Microfuge at 14,000 rpm for 3 minutes at room temp. Try to remove as much buffer as possible after each wash without aspirating the Staph A cells. Efficient washing is critical to reduce background. 15. After the last wash, Microfuge and remove the last traces of buffer. Elute antibody/protein/DNA complexes by adding 150 uls of IP elution buffer. Shake on vortexer for at least 15 minutes at setting "vortex 3". Microfuge at 14,000 rpm for 3 minutes. Transfer supernatants to clean tubes. Repeat and combine.

Although ChIP kits have been used mostly in mammalian samples, it is possible that some antibodies will recognize plant Histone due to high sequence homology. However, you may need to alter the extraction/lysis buffer. Plant preparation, lysis buffer, and sonication strategies, to shear plant Histones down to about three nucleosomes, would have to be worked out. Please see the following references on Histone acetylation and synthesis in plants: Wang, et al.(2002), The Plant Journal 32 (5), 831-843; Waterborg, J.H. Biochem. Cell Biol. 80: 363-378, 2002; Waterborg, J.H. and Kapros, Cell Biol. 80: 279-293, 2002; Waterborg, J.H. JBC, 268: 4912-4917, 1993; Waterborg. J.H. JBC, 268: 4918-4921, 1993; Waterborg, J.H. JBC, 265: 17157-17161, 1990; Waterborg, J.H. Plant Physiol. 96: 453-458, 1991; Waterborg, J.H. Plant Mol. Biol. 18: 181-187, 1992; Waterborg, J.H. Biotechnology Quarterly 9: 12-13, 1992; Waterborg, J.H. Biochemistry 31: 6211-6219, 1992.

Nothing really. Many customers perceived that only 'ChIP grade' antibodies would work in ChIP so we have eliminated this term. 'ChIP grade' antibodies were designed specifically for ChIP, but also work well in other applications. It is best to consult the list of tested applications for each product to determine if it is suitable for use in ChIP.

For ChIP to be successful, it requires that the epitope recognized by the antibody that you are employing be available after cross-linking and not buried in the protein complex. Antibodies also have to be of very high avidity so that the interaction with the protein will survive the washing steps. It is also important that the protein you are trying to IP will cross-link efficiently to the chromatin.

Yes, but the antibodies must be very good and generally you will want to increase the fixation times to insure maximal cross-linking.

Any process that uses DNA as a template (replication, transcription, mitosis, etc.) needs to contend with its organization into chromatin (the protein-DNA complex that is present within the nucleus of eukaryotic cells).

To insure good resolution for ChIP. If your average fragment size is greater than 1000 bp, you could be pulling down DNA that contains your target sequence for PCR but the protein of interest may be over 700 nucleotides distant from your target.

Primer length should be 24 nucleotides; they should have 50% GC content, and a Tm of 60°C. Don't try to amplify anything larger than 600-800 nucleotides. Attempt to stay away from sequences that are not unique within the genome.

GAPDH is a good control if you are using an antibody specific for acetylated Histones.

Salmon Sperm is used to reduce the non-specific interaction of chromatin DNA with the agarose. It is unlikely that people will be performing ChIP from salmon tissues, so the DNA shouldn't amplify with your PCR primers due to cross-hybridization.

ChIP control: use Anti-acetyl H3 primary antibody and PCR for the GAPDH gene promoter. This will ensure that each step of the procedure is working. PCR amplification: Control for PCR amplification using primers designed against a sequence that would not be enriched by your chromatin IP. Liner Range PCR controls: Ensure that PCR amplification is in the linear range by setting up each reaction at different dilutions of DNA for various amplification cycle numbers, and select the final PCR conditions accordingly. The assays are typically done in duplicate or triplicate. The average fragment size after sonication is ~500 bp (Kondo, et al. Molecular and Cellular Biology, January 2003, p. 206-215, Vol. 23, No. 1) Treatment controls: 1) ChIP analysis of a transcribed region of the gene of interest which is >40 kb away from the promoter you are looking at. This may reveal that the activation level (e.g., acetylation level) may be very low or more importantly, not affected by your treatment. 2) Control for specificity of an induced local Histone hyperacetylation, you could analyze the acetylation level of another promoter (Sachs, et al. Proc. Natl. Acad. Sci. USA 97:2000, 13138-13143). No primary antibody control: This is the control in which you run the ChIP assay but don't add the primary immunoprecipitating antibody. It will ensure that you are not seeing sequences that bind non-specifically to the beads and that the recognition of your protein by the antibody you are using is required for enrichment of the target sequence Negative antibody control: A normal serum, normal IgG, or an antibody to a distant protein (all from the same species) is a good negative antibody control. The best control if using a polyclonal antibody is pre-immune antiserum of the animal that has been immunized.

Input DNA is DNA obtained from chromatin that has been cross-link reversed similar to your samples. It is a control for PCR effectiveness. Output DNA is the DNA from each of your ChIP experiments.

To eliminate banding in your negative controls you can do several things. A) Pre-clear the 2ml diluted cell pellet suspension with 80 microliters of Salmon Sperm DNA/Protein A Agarose-50% Slurry for 30 minutes at 4ºC with agitation. You could try to preclear the lysate longer or with more clearings. B) Titrate your input DNA, to see when the bands in the NFA disappear. C) Use an alternative lysis procedure: Resuspend cell pellet in 200 microliters of 5mM Pipes pH 8.0, 85mM KCl, 0.5% NP40 containing protease inhibitors. Place on ice for 10 minutes. Pellet by centrifugation (5 minutes at 5000 rpm). Resuspend pellet in 200 microliters of 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1 containing protease inhibitors. Incubate on ice for 10 minutes. D) Block the Salmon Sperm DNA Agarose prior to use in 1-5% BSA and Chip dilution buffer (mix at room temperature for 30 minutes). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernatant. Wash once in ChIP assay buffer and continue.

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).

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.

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.

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 method is described in Suka et al (2001) Molec. Cell, 8: 473-479.

Please see the manual for The EZ ChIP Kit (Catalog #17-371) for more information.

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.

You should resuspend your pellet in water and not TE as the EDTA found in the TE may interfere with PCR.