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Thermo Scientific™ Pierce™ Agarose ChIP Kit

Catalog No. PI26156
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30 Reaction Kit
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PI26156 30 Reaction Kit
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Catalog No. PI26156 Supplier Thermo Scientific™ Supplier No. LSG26156
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Description

Chromatin immunoprecipitation (chromatin IP, ChIP) assay kit containing all reagents, agarose affinity beads, and a simple, fast, reliable protocol.

Thermo Scientific™ Pierce Agarose ChIP Kit provides a complete set of reagents and a simple, fast and reproducible protocol to perform chromatin immunoprecipitation (ChIP) assays.

This chromatin IP kit contains sufficient reagents to perform 30 ChIP assays with appropriate controls using an optimized protocol and a convenient spin-column format. The kit includes a complete Chromatin Prep Module (also available separately) to efficiently extract and solubilize crosslinked DNA-protein interaction complexes from cells, plus an immunoprecipitation module that contains high-capacity, ChIP-grade Protein A/G agarose.

Highlights:

  • Simple and streamlined ChIP assay protocol (< 8 hours)
  • Highly efficient isolation and lysis of nuclei
  • Easy and reproducible enzymatic digestion
  • Low-background and high-capacity Protein A/G agarose resin
  • Highly specific positive control reagents included: RNA polymerase II antibody and GAPDH PCR primers
  • Fast and reproducible spin-column format; columns and collection tubes included
  • High-recovery DNA purification
  • Choose a complete ChIP assay kit or the separate chromatin preparation module

Includes:

Kit contains the Chromatin Prep Module (Part No. 26158; 11 components) for chromatin extraction and a complete set of immunoprecipitation reagents (15 components) for IP experiments with controls

Specifications

Binding Capacity >50 mg Human IgG/mL resin binding capacity
Capacity (Metric) ≥50 mg Human IgG per mL Resin Binding
Description Pierce Agarose ChIP Kit
Content And Storage Sufficient For: 30 complete chromatin IP assay experiments• ChIP Grade Protein A/G Plus Agarose, 0.65 mL (store at 4°C)
• IP Dilution/Wash Buffer (5X), 11 mL (store at 4°C)
• IP Wash Buffer 3 (5X), 4.5 mL (store at 4°C)
• IP Elution Buffer (2X), 4.5 mL (store at 4°C)
• Column Accessory Pack, 3 packs (store at room temperature or 4°C)
• Microcentrifuge Tubes (1.5mL), 75 tubes (store at room temperature or 4°C)
• DNA Clean-Up Column, 40 columns (store at room temperature or 4°C)
• DNA Column Binding Solution, 30 mL (store at room temperature or 4°C)
• DNA Column Wash Solution, 6 mL (store at room temperature or 4°C)
• pH Indicator, 0.8 mL (store at room temperature or 4°C)
• DNA Column Elution Solution, 5 mL (store at room temperature or 4°C)
• Anti-RNA Polymerase II Antibody, 25 μL (store at -20°C)
• Normal Rabbit IgG (1 mg/mL), 10 μL (store at -20°C)
• ChIP Positive Control Primers (GAPDH promoter, Human-specific), 100 μL (store at -20°C)
• Pierce Chromatin Prep Module (Part No. 26158) (store Proteinase K and Micrococcal Nuclease at -20°C. Store all other components at 4°C).
Product Line Pierce
Quantity 30 Reaction Kit
Type ChIP Kit
Sufficient For 30 Reactions
Format Kit

Frequently Asked Questions (FAQs)

I am having trouble with leaking columns while using the Pierce Agarose ChIP Kit (Cat. No. 26156). How can I resolve this issue?

It is common to experience leaking when using the columns, even when the plug is pushed in very tightly. We recommend wrapping the plug with parafilm to prevent leaking.
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 Agarose ChIP Kit, what can cause no or low PCR signal in the experimental IP samples?

Here are possible causes and solutions:

- Insufficient amount of sample DNA added to the PCR reaction: Add more sample DNA to the PCR reaction.
- 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.

Note: Increasing the stringency of the nuclear lysis is not recommended unless there is no signal or low signal-to-noise in the IP. Excess sample can result in high background decreasing the signal of the specific signal.
With my Agarose ChIP Kit, what can cause PCR signal of the positive and negative control IP samples to be equivalent?

Here are possible causes and solutions:

- Insufficient washing of the IP complex: Include an additional wash with Buffers 2 and 3.
- 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.
With my Agarose 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.
- Incomplete elution from the Protein A/G agarose resin: Perform elution at 65 degrees C and increase frequency of mixing.
With my Agarose 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.

Note: Increasing the stringency of the nuclear lysis is not recommended unless there is no signal or low signal-to-noise in the IP. Excess sample can result in high background, decreasing the signal of the specific signal.
I used the Agarose ChIP Kit and did not recover chromatin from the nucleus. What happened?

This could be because your cell type requires more stringent handling to better lyse and release chromatin from the nucleus. Here are some suggestions:

  • Increase incubation with nuclei lysis buffer to 30 minutes and vortex for 30 seconds every 5 minutes.
  • Following the nuclear lysis step, sonicate the sample for 60 seconds using a 1/8 probe. Perform on wet ice in 3 pulses of 20 seconds with 30 second pauses between.
  • Following the nuclear lysis step, dounce the sample 20 times in a glass dounce homogenizer.
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 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.