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Thermo Scientific™ Pierce™ Magnetic ChIP Kit

Catalog No. PI26157
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PI26157 30 reactions
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Catalog No. PI26157 Supplier Thermo Scientific™ Supplier No. 26157
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Description

Efficiently isolate chromatin-bound DNA by immunoprecipitation (chromatin IP) for subsequent quantitation by PCR using Thermo Scientific™ Pierce Magnetic ChIP Kit.

Efficiently isolate chromatin-bound DNA by immunoprecipitation (chromatin IP) for subsequent quantitation by PCR using Thermo Scientific™ Pierce Magnetic ChIP Kit.

Additional Information and Online Ordering

The Pierce Magnetic ChIP Kit contains sufficient reagents to perform 30 chromatin immunoprecipitation (ChIP) assays with appropriate controls using an optimized protocol. The kit includes reagents for cell lysis, capture of protein-DNA complexes, reversal of crosslinking, and DNA isolation. The blocked Pierce Protein A/G Magnetic Beads provide high binding capacity, low non-specific background, and work with many antibody species. These beads can be used manually with a magnetic stand or with automated platforms, such as the Thermo Scientific KingFisher Instruments. ChIP-validated and quality-guaranteed antibodies are also available for use with the Pierce Magnetic ChIP Kit.

Highlights:

  • Rapid – obtain purified DNA that is ready for quantitative PCR in about 8 hours
  • Efficient and reproducible – micrococcal nuclease digestion and nuclear lysis are highly optimized
  • Sensitive – obtain results with as few as 10,000 cells (1 x 104)
  • Low background – Pierce Protein A/G Magnetic Beads are blocked in a non-DNA-containing reagent to minimize background

  • Complete – optimized positive control reagents are included: RNA polymerase II antibody and GAPDH promoter PCR primers

Includes:

Kit contains reagents for chromatin preparation, IP, DNA purification, and positive controls (antibody and primers)

Requires:

In vivo crosslinker (such as formaldehyde), micro-tip sonicator (such as Misonix™ Sonicator 3000), ChIP qualified antibody of choice, PCR primers for DNA sequence of interest, PCR master mix (containing a dye such as SYBR™ Green, if qPCR is desired), and a qPCR instrument

Recommended for:

  • Determine sites of specific protein-DNA interactions on genomic DNA by PCR or ChIP-Seq
  • Monitor the effects of histone modifications or chemical agents on DNA binding

Specifications

Capacity (Metric) 700 μL
Description Pierce Magnetic ChIP Kit
Kit Contents 4 x 106 cells/reaction
Content And Storage Slurry: 10 mg/mL
• Glycine Solution (10X), 15 mL; store at room temperature
• PBS (20X), 15 mL; store at 4°C
• Halt™ Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X), 1 mL; store at 4°C
• Membrane Extraction Buffer, 10 mL; store at 4°C
• MNase Digestion Buffer, 10 mL; store at 4°C
• DTT (7.7 mg), Lyophilized, 2 vials; store at room temperature or 4°C
• MNase Stop Solution, 1 mL; store at 4°C
• ChIP-Grade Protein A/G Magnetic Beads, 0.7mL; store at 4°C
• IP Dilution/Wash Buffer (5X), 40 mL, store at 4°C
• Sodium Chloride (5M), 6 mL; store at 4°C
• IP Elution Buffer (2X), 4.5 mL; store at 4°C
• Microcentrifuge Tubes, 1.5 mL, 75 tubes; store at room temperature or 4°C
• DNA Clean-Up Column and Reagents, 40 purifications; store at room temperature or 4°C
• DNA Clean-Up Column, 40 columns
• DNA Column Binding Solution, 30 mL
• DNA Column Wash Solution, 6 mL
• pH Indicator, 0.8 mL
• DNA Column Elution Solution, 5 mL
• Micrococcal Nuclease (ChIP Grade) (10U/μL), 50 μL; store at -20°C
• Anti-RNA Polymerase II Antibody, 25 μL; store at -20°C
• Normal Rabbit IgG (1mg/mL), 10 μL; store at -20°C
• Proteinase K (20mg/mL), 0.25 mL; store at -20°C
• ChIP Positive Control Primers (GAPDH promoter, Human-specific), 100 μL; store at -20°C
Product Line Pierce
Quantity 30 reactions
Type ChIP Kit
Sufficient For 30 Reactions
Format Kit

Frequently Asked Questions (FAQs)

With my ChIP assay (Agarose ChIP Kit/Magnetic ChIP Kit), the qPCR signal (cycle threshold) from my positive and negative controls show no difference or are very close. Why is this?

-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.
With my Magnetic ChIP Kit, what can cause no or low PCR signal in the experimental IP samples?

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.
With my Magnetic ChIP Kit, what can cause PCR signal of the positive and negative control IP samples to be equivalent?

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.
With my Magnetic ChIP Kit, what can cause no or low PCR signal in the positive control IP samples?

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).
With my Magnetic ChIP Kit, what can cause no or low PCR signal in the total input control samples?

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

My chromatin fragments are too small (< 200 base pairs) after using the Agarose ChIP Kit/Magnetic ChIP Kit. Why is this?

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).
My chromatin fragments are too large (> 1000 base pairs) after using the Agarose ChIP Kit/Magnetic ChIP Kit. Why is this?

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)

Do I have to use formaldehyde crosslinking with the Agarose ChIP Kit (Cat. No. 26156) and Magnetic ChIP Kit (Cat. No. 26157)?

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.
What is a qPCR cycle threshold (Ct) and what are typical Ct results when performing ChIP assays?

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.
Why do you prefer enzymatic digestion of chromatin over traditional sonication?

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.
Can I use the Agarose ChIP kit (Cat. No. 26156) for ChIP-seq?

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.
What is the difference between the Agarose ChIP Kit (Cat. No. 26156) and the Magnetic ChIP Kit (Cat. No. 26157)?

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.
I am getting equivalent PCR signal from positive and negative control IP samples. Why is this?

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.

I am not getting PCR signal in the experimental IP samples, while the results from positive and negative control IPs are as expected. What happened?

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.

I am not getting any PCR signal with the positive control IP samples. Do you have some tips?

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.

I am doing chromatin analysis and am not getting PCR signal in the total input control samples. What should I do?

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.

What is the optimal fragment size for a ChIP reaction?

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.

What is the purpose of sonication in the Thermo Scientific Magnetic ChIP Kit, when Micrococcal Nuclease is used to fragment DNA?

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.

What controls should I use for ChIP?

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.

Which method do you recommend to shear DNA fragments for ChIP?

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.

What is the expected yield in ChIP reactions?

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.

What is the recommended amount of starting material when working with your ChIP kits?

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.

For Research Use Only. Not for use in diagnostic procedures.