Chromatin IP Frequently Asked Questions

Answers

Does the number of cell I harvest influence the amount of micrococcal nuclease I use in the digest?

The number of cells used for harvesting chromatin is very important. We typically harvest 4x10⁷ cells in a single 1.0 ml preparation of chromatin and digest with 5 μl of micrococcal nuclease. The ratio of cell number 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 types, we find that a ratio of 1x10⁷ cells to 1.25 μl of micrococcal nuclease reproducibly digests chromatin to the appropriate size fragments.

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?

As described in Section B of the SimpleChIP^® 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 types. If there are problems with chromatin appearance, please refer to Appendix A in the protocol.

*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 you are using. Add less nuclease or increase the number of cells in the digest (see Appendix A for optimization).

How much chromatin do I need per IP?

We typically use 4x10⁶ cell equivalents, or 10 to 20 μg of chromatin, per IP for all protein targets; however, as little as 1x10⁶ cell equivalents, or 2.5 to 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^® Enzymatic Chromatin IP Kit 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. 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^® Enzymatic Chromatin IP Kit. 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 C_T 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 C_T 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 (ie. 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 (ie. 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 (ie. transcription factors and co-factors) to as high as 40 to 50 percent total input chromatin (ie. acetylated and methylated histones). Using Normal Rabbit IgG #2729, our background levels with magnetic and agarose beads typically range from 0.1 to 0.05 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.

Can I use SimpleChIP^® Enzymatic Chromatin IP Kits #9002 and #9003 with tissue samples?

We are currently testing our SimpleChIP^® Enzymatic Chromatin IP Kits on cross-linked tissue samples. We have very promising data from several collaborators, and we are currently developing a protocol. If a customer is interested in performing ChIP on tissue samples, he/she can contact us and we will provide a preliminary protocol that he/she can use in conjunction with the SimpleChIP^® Kits. We hope to optimize the protocol and include it with kits in the near future.

If the customer has any questions, he/she can contact us directly at CST_ChIP@cellsignal.com.