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View additional product information for Pierce™ Magnetic ChIP Kit - FAQs (26157)
22 product FAQs found
-Verify that your specific antibody (if not using the kit-provided RNA polymerase II antibody) is validated for IP. Ideally, a ChIP validated antibody is the best, but an antibody for IP has a good chance of working in ChIP.
-Ensure that your chromatin is properly digested (see Appendix A in the manual). Too much digestion as well as too little digestion will affect the success of the ChIP reaction.
-Ensure that all the chromatin has been released from the nuclei. When following the Magnetic ChIP kit instructions, MNase digestion of 4x106 cells followed by sonication to lyse the nuclei, yields about 20-50 µg for the IP. This same sequence can be used with the Agarose ChIP Kit as well. It is recommended that you start with 2–4 x 106 cells per ChIP reaction. Once a successful ChIP has been run at this cell number, it is possible to decrease the cell amount empirically. We have seen good results using as little at 10,000 cells, but this entirely depends on the cell line, target, and antibody.
-Ensure that enough DNA was used for qPCR. Typically, 30-80 ng of DNA is a good range.
Here are possible causes and solutions:
- Insufficient amount of antibody added to the IP: Add more antibody to the IP.
- Antibody did not function in an IP: Verify that the antibody is qualified for CHIP or IP applications and has been handled and stored properly.
Here are possible causes and solutions:
- Excess chromatin or antibody added to the IP: Add less chromatin or antibody.
- PCR amplification was measured outside the linear range of amplification: Decrease the number of amplification cycles used in the PCR reaction.
- Insufficient amount of sample DNA added to the PCR reaction: Add more sample DNA to the PCR reaction.
Here are possible causes and solutions:
- Insufficient chromatin amount in the IP reaction: Use at least 25 µg of chromatin for each IP.
- Insufficient antibody incubation time: Incubate antibody overnight.
- Nuclei not fully lysed: Monitor sonication of nuclei by microscope to ensure full lysis.
- Low-abundance target: Add more chromatin or magnetic beads (30 µL).
Here are possible causes and solutions:
- PCR amplification conditions were not fully optimized: Optimize PCR conditions using samples known to contain the target amplicon; Check primer design
- Insufficient amount of sample DNA added to the PCR reaction: Increase the amount of sample DNA added to the PCR reaction
- Nuclei not fully lysed: Monitor sonication of nuclei by microscope to ensure full lysis
This indicates that the cell to Micrococcal Nuclease (MNase) ratio was too low. Decrease the amount of MNase or increase the cell number (refer to the MNase digestion optimization protocol in Appendix A of the manual).
Here are possible causes and solutions:
- Crosslinking time was too long: Shorten crosslinking time
- Cell to Micrococcal Nuclease (MNase) ratio was too high: Increase amount of MNase or decrease cell number (refer to the MNase digestion optimization protocol in Appendix A of the manual)
These kits were designed using formaldehyde as a crosslinker. The lysing, washing, and elution conditions have all been optimized for formaldehyde crosslinking. These steps are not optimal for native ChIP (no crosslinking,) or for other crosslinkers, such as EGS.
The cycle threshold is the PCR cycle number where the fluorescent signal exceeds the background threshold level. This number is determined by your qPCR instrument and software.
A typical signal from the rabbit IgG IP is usually ≥30 cycles while the typical signal from a positive control antibody IP (such as the RNA-polymerase antibody provided in the kit) is ~25 cycles.
To be considered significant, a Ct difference of at least 3 is needed between the negative control (rabbit IgG) and your specific antibody.
The advantages of enzymatic digestion include reproducibility of digestion, control of the reaction, and easy titration of the enzyme for each specific cell type. Our ChIP kits include a specially titrated and tested micrococcal nuclease that digests the DNA, minimizing variable results caused by the traditional method of sonication.
The Agarose ChIP Kit is not appropriate for use with ChIP-seq applications; however, the Magnetic ChIP Kit (Cat. No. 26157) can be used for ChIP-seq applications as the magnetic beads are blocked with a non-DNA containing buffer.
There are a few major differences between our agarose and magnetic ChIP kits. One is, of course, the beads. Both beads are blocked with different reagents. The magnetic beads are amenable to ChIP-seq as well as automation for the washing and elution steps. The second major difference is the chromatin preparation. Both kits use MNase to shear the chromatin into the appropriate sized fragments. The agarose kit uses a high-salt buffer to leak the nuclear contents after shearing, while the magnetic kit uses a quick sonication step to mechanically disrupt the crosslinked nuclei. The high-salt extraction can be very effective, however, if trouble is seen, the mechanical disruption can greatly improve results. The third major difference is the wash buffers. Each kit was optimized for its specific bead matrix (agarose or magnetic beads) to minimize background.
There might be excess chromatin or antibody added to the IP, or insufficient amount of DNA template added into the PCR reaction. Also your PCR conditions might need optimizing. Try decreasing the number of amplification cycles in PCR. Finally, it is ideal to have duplicate or triplicate runs for each IP to identify any issues, like human or product errors.
The most possible cause is the antibody does not function in IP. Not all antibodies used for western blotting will work well in ChIP. You need to verify the antibody is qualified for ChIP or IP applications. And try adding more antibody to the IP reaction and more DNA template to the PCR.
This is likely to be caused by insufficient chromatin amount in the IP reaction or insufficient antibody incubation time. We also recommend optimizing the crosslinking condition, and monitoring sonication of nuclei by microscope to ensure complete lysis.
First make sure that the PCR condition is fully optimized and check the primer design. Then try increasing the amount of template DNA added to the PCR reaction. Finally, evaluate sonication of nuclei by microscope to ensure complete lysis.
Shearing of the chromatin into appropriate fragments is required to ensure optimal ChIP results. Ideal shearing should yield DNA fragments between 200 and 1000 bp in length. ~200-500 bp fragments are usually used in downstream qPCR analysis and ~100-300 bp fragments are used in massive parallel DNA sequencing downstream analysis.
Sonication is used to break open the nucleus, allowing the digested DNA fragments to be released and extracted. In the Thermo Scientific Agarose ChIP Kit, a high-salt method is used to extract fragmented DNA. In principle, both methods work well for this purpose but sonication is easier.
We recommend the following controls:
- Negative control antibody: Either do not use a primary antibody, or use the normal rabbit IgG or mouse IgG isotype control.
- Positive control antibody: This control ensures that each step of the procedure is working. For example, we observe consistent enrichment of heterochromatin markers such as H3-K9Me3 at the satellite repeat locus (SAT-2).
- Negative control PCR primer: This control is designed against a sequence that would not be enriched by your chromatin IP procedure.
- Input DNA control: Input DNA is DNA obtained from chromatin that has not been immunoprecipitated and has been reversed crosslinked similar to your samples. It is a control for PCR effectiveness and utilized in ChIP sequencing data analysis.
Both sonication and enzyme digestion are widely used for ChIP. Micrococcal nuclease results in greater reproducibility and allows better control of the fragment sizes. However, it can take more optimization and the enzyme can be compromised and fail to work properly.
Many factors influence yield, including cell type, target protein, antibody, etc. Generally speaking, anywhere between 0.5 ng to >10 ng can be obtained per ChIP reaction. Yields are usually too low to be measured using a NanoDrop spectrophotometer.
For each ChIP reaction, the MAGnify Chromatin Immunoprecipitation (ChIP) System recommends using 10,000-300,000 cells or 0.167 - 5 mg of tissue. The Thermo Scientific Agarose ChIP Kit and Thermo Scientific Magnetic ChIP Kit recommend 2 - 4 x 10E6 cells per ChIP reaction. To ensure consistency and decrease experimental variability, we recommend preparing a common chromatin batch suitable for multiple ChIP experiments.