Questions

Answers

What is chromatin immunoprecipitation (ChIP)?
The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. This assay can be used to identify multiple proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome associated with a particular protein. In addition, the ChIP assay can be used to define the spatial and temporal relationship of a particular protein-DNA interaction. For example, the ChIP assay can be used to determine the specific order of recruitment of various protein factors to a gene promoter or to “measure” the relative amount of a particular histone modification across an entire gene locus during gene activation. In addition to histone proteins, the ChIP assay can also be used to analyze binding of transcription factors, transcription co-factors, DNA replication factors, and DNA repair proteins. Please see our ChIP Assay Overview for more details.
Can I use SimpleChIP® Enzymatic Chromatin IP Kits with tissue samples?
Yes. The original SimpleChIP® Enzymatic Chromatin IP Kits (#9002 and #9003) contain a detailed protocol and reagents needed to prepare chromatin and perform immunoprecipitations from cultured cells. The new SimpleChIP® Plus Enzymatic Chromatin IP Kits (#9004 and #9005) were developed to work with both cultured cells and tissue samples, and contain a detailed protocol for cross-linking, preparing chromatin, and performing immunoprecipitations from both cells and tissue samples. In addition, the SimpleChIP® Plus protocol is readily scalable, allowing the user to quickly determine the amount of reagents to use at each step of the protocol based on the number of immunoprecipitations they are performing. All SimpleChIP® Kits are available with either Protein G Agarose or Protein G Magnetic beads and contain all buffers and reagents needed to perform up to 30 ChIP assays. In addition, each kit includes a Troubleshooting Guide discussing expected chromatin yield and optimization of the chromatin digest.
Can I use SimpleChIP® Enzymatic Chromatin IP Kits for ChIP-Sequencing?
The SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 and SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 can both be used for ChIP-Seq experiments. These kits contain Protein G Magnetic beads, which do not require a DNA blocking agent. This is critical for ChIP-Seq because any carryover of the blocking DNA would contaminate downstream sequencing reads.
Why is enzymatic digestion of chromatin better than sonication-based fragmentation?
While sonication has been the traditional method used for fragmenting chromatin, many problems can occur due to variability in sonication power and the emulsification of the chromatin sample during sonication. While under-sonication can lead to incomplete chromatin fragmentation, over-sonication or emulsification of the sample can result in the loss of antibody epitopes due to protein denaturation and degradation. We performed a ChIP Methods Comparison and found that enzymatic digestion of chromatin from cultured cells is much milder than sonication and leads to a dramatic increase in immunoprecipitation efficiencies in the ChIP assay. A Comparison of Tissue-Based ChIP Kits also shows increased immunoprecipitation efficiencies using digested chromatin from tissue samples.
How does the amount of cells or tissue I harvest influence the amount of micrococcal nuclease I use in the digest?
Measuring the number of cells or amount of tissue used to prepare the chromatin is very important. We recommend harvesting 4x106 cells or 25 mg of tissue for each planned chromatin immunoprecipitation reaction. Tissue samples are cross-linked and disaggregated into single-cell suspensions prior to enzymatic digestion of the chromatin. The ratio of the number of cells to volume of micrococcal nuclease added to the chromatin digestion is critical for fragmenting the chromatin to the appropriate size (150 to 1000 base pairs). Although this ratio will differ slightly with different cell or tissue types, we find that a ratio of 4x106 cells (or 25 mg of tissue) to 0.5 μl of micrococcal nuclease reproducibly digests chromatin to the appropriate size fragments. Each SimpleChIP® Kit provides a Troubleshooting Guide for optimization of the chromatin digestion using cells or tissue.
Why do I need to sonicate if I use micrococcal nuclease to digest the chromatin?
Incubation in Buffers A and B does not completely lyse the cell and nuclear membranes of formaldehyde cross-linked cells. Instead, it only permeabilizes the cell, allowing micrococcal nuclease to enter and digest the chromatin. Therefore, a small amount of sonication is required to release the chromatin into solution. Sonication does not further fragment the chromatin.
What should my chromatin look like on the agarose gel?
1% agarose gel stained with ethidium bromide.

As described in Section B of the SimpleChIP® protocol and Section IV of the SimpleChIP® Plus protocol, we analyze a sample of the chromatin after digestion and prior to performing immunoprecipitations. Please see the agarose gel to the right for ideal chromatin appearance on a 1% agarose gel stained with ethidium bromide. DNA marker is in lane 1, while purified chromatin DNA is in lane 2. The chromatin DNA should be sheared into mono-, di-, tri-, tetra- and penta-nucleosome units (150 to 1000 base pairs in length). We determine chromatin DNA concentration based on OD260, and typically observe 125–250 μg/ml with various cell and tissue types. If there are problems with chromatin appearance, please refer to the appendices of each protocol for optimization of the chromatin digestion.

*Note: If you observe only a single band around 150 bp (mono-nucleosome), the chromatin is over-digested. You are adding too much nuclease for the number of cells or amount of tissue you are using. Add less nuclease or increase the number of cells or amount of tissue in the digest (see appendices in protocols for optimization).

How much chromatin do I need per IP?
We typically start with 4x106 cells or 25 mg of tissue sample per immunoprecipitation (IP) for all protein targets. This typically translates to 10–20 μg of chromatin per IP. However, as little as 1x106 cell equivalents, or 2.5–5 μg of chromatin, will work for histone IPs. When setting up IPs, the chromatin is typically diluted into 1X ChIP Buffer to make a 500 μl IP reaction, which is incubated with antibody overnight. However, one does NOT need to dilute the chromatin when setting up the IP. The user can set up his/her IPs using undiluted chromatin in any desired volume.
What are the correct antibody conditions?

If the antibody has been validated for ChIP at Cell Signaling Technology (CST), refer to the antibody product data sheet for the correct amount of antibody to use in an IP. The dilution to use with the SimpleChIP® Kits is provided under “Recommended Antibody Dilutions”, while the exact volume of antibody and amount of chromatin used for validation experiments is provided in the “Chromatin IP” figure legend on the antibody product data sheet. When validating an antibody at CST, we always titrate the antibody to determine the optimal dilution for use with 4x106 cells (10–20 μg of chromatin).

If the antibody has not been validated at CST, we cannot guarantee the antibody’s performance in the ChIP assay. If one wants to try the antibody in the ChIP assay, we recommend only using antibodies that have previously been validated for normal IP, and we recommend using 2–5 μg of antibody per chromatin IP reaction. We would like to receive feedback from customers if they have success with a CST antibody that we have not yet validated.

What are the advantages/disadvantages of Protein G Agarose versus Protein G Magnetic Beads?

Both beads perform identically in the SimpleChIP® Kits. We have done side-by-side comparisons showing the same minimal background signal and specific binding using both beads. Agarose beads are the traditional beads used for IPs. However, magnetic beads, though slightly more expensive, are easier to use. The beads adhere to the side of the tube when aspirating the supernatant, resulting in no loss of material during aspiration of the wash buffers and more complete washes since one can aspirate all of the supernatant from the beads. They do not require a centrifuge, however, a 6-Tube Magnetic Separation Rack #7017 is required.

The magnetic beads can be used for ChIP-Sequencing experiments, as they are not blocked with DNA. Agarose beads are blocked with sonicated salmon sperm DNA to reduce background signal. Any carryover of the blocking DNA will contribute to sequence reads when performing the high throughput sequencing.

What can I expect for PCR results?

At CST, we validate our antibodies using quantitative real-time PCR and a 4 point, 5-fold dilution series, starting with 2% of the total input chromatin. We use this dilution series to create a standard curve of CT values for each input chromatin qPCR sample (2%, 0.4%, 0.08%, 0.016%). This allows us to calculate the DNA enrichment in each IP sample by converting the measured CT value for each IP sample qPCR reaction to a value represented as a percent of the total input chromatin.

If you use the control Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620 and the control SimpleChIP® RPL30 primers provided in the kits, you should see an enrichment of the RPL30 promoter between 2 to 4 percent of the total input chromatin. Background enrichment with Normal Rabbit IgG #2729 should be less than 0.1 percent of the total input chromatin.

We define a ‘positive’ ChIP result as an antibody enrichment of a specific genomic locus (i.e. binding of a transcription factor to its target promoter) that is at least 4 fold greater than enrichment of a non-specific locus with the same antibody (i.e. binding of the same transcription factor to a non-target promoter), and at least 5 to 10 fold greater than enrichment of the specific locus with Normal Rabbit IgG #2729. Positive ChIP enrichments can range from as little as 0.5 percent total input chromatin (i.e. transcription factors and co-factors) to as high as 40 to 50 percent total input chromatin (i.e. acetylated and methylated histones). Using Normal Rabbit IgG #2729, our background levels with magnetic and agarose beads typically range from 0.05 to 0.1 percent of the total input chromatin.

PCR results will vary based on PCR primer sets and antibodies used. Experiments should be designed with appropriate positive and negative controls to ensure that the PCR reaction is amplifying properly and any signal obtained is real.

How do I know if my chromatin IP experiment worked?

SimpleChIP® Control PCR Primers provide the perfect positive and negative controls for your ChIP experiments and ChIP antibodies. If you use the positive control Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620 and the positive control SimpleChIP® RPL30 primers provided in the kits, you should see an enrichment of the RPL30 promoter between 2 to 4 percent of the total input chromatin. Background enrichment of the RPL30 promoter with negative control Normal Rabbit IgG #2729 should be less than 0.1 percent of the total input chromatin. In addition, since histone H3 is a core component of chromatin in the cell and is bound to most DNA sequences throughout the genome, the Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620 can be used as a positive control primer set for virtually any DNA locus studied.

We sell both positive and negative control primer sets for all of our ChIP-validated antibodies. These are the same primer sets we use for validation of our antibodies in ChIP. Positive control primer sets amplify gene loci that have been shown to contain the specified histone modification or to be bound by the specified transcription factor, while negative control primer sets amplify regions that do not contain the specified modification or bound factor. Please note that histone modifications and transcription factors found at a given gene locus can differ based on cell context, so some control primer sets may be specific to a given cell type and/or treatment, depending on the histone modification or transcription factor being studied. However, other control primer sets are more universal and can be used as controls for multiple antibodies in multiple cell types. For example, RPL30 and GAPDH are housekeeping genes that are active in most cell types. Therefore, the RPL30 and GAPDH control primer sets can be used as positive controls for histone modifications associated with transcriptional activation in most cell types. Similarly, the a satellite repeat control primer set amplifies a region of constitutive heterochromatin and can be used as a negative control for histone modifications and transcription factors associated with transcriptional activation in any cell type.

Can I purchase individual SimpleChIP® Enzymatic Chromatin IP Kit components?
The SimpleChIP® Kits contain enough of each reagent to support up to 30 chromatin immunoprecipitations. Currently, these kits can only be purchased in their entirety. However, if you are in need of a specific kit component, please contact us and we may be able to support your needs with a custom order or provide an alternative source.

For any additional questions, please contact our ChIP group directly at CST_ChIP@cellsignal.com

Christopher Fry, PhD
Development Scientist

Curtis Desilets
Chromatin IP Specialist

Stephen Manning
Product Scientist


posted October 2010

revised October 2012