Pierce™ Magnetic ChIP Kit
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Thermo Scientific™

Pierce™ Magnetic ChIP Kit

The Thermo Scientific Pierce Magnetic ChIP Kit provides a convenient method for efficient isolation of chromatin-bound DNA by immunoprecipitation (chromatinRead more
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Catalog number 26157
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802.00
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Price (USD)
802.00
Each
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The Thermo Scientific Pierce Magnetic ChIP Kit provides a convenient method for efficient isolation of chromatin-bound DNA by immunoprecipitation (chromatin IP) for subsequent quantitation by PCR.

Features of the Magnetic ChIP Kit:

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 10^4)
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

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.

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

Applications:
• 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

A successful ChIP assay requires a number of critical steps (crosslinking, chromatin preparation, immunoprecipitation) prior to detecting the target genomic DNA. Individually, each step in the ChIP protocol can be time-consuming to optimize. The Thermo Scientific Pierce Magnetic ChIP Kit simplifies the ChIP process, enabling accurate and reproducible results.

Protein-DNA complexes are first crosslinked in vivo with formaldehyde. The kit contains reagents to lyse cells and extract and solubilize the crosslinked complexes. The complexes are then incubated with a specific antibody and isolated using Pierce Protein A/G Magnetic Beads. After reversing crosslinks and digesting protein, the resulting DNA fragments are purified and are then ready for standard or quantitative PCR.

More Product Data
Analysis of androgen-dependent and -independent regulation of transcriptional activity

Related Products
Pierce™ Magnetic ChIP Kit
Pierce™ ChIP-grade Protein A/G Magnetic Beads
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Capacity (Metric)4 x 106 cells/reaction
DescriptionPierce Magnetic ChIP Kit
Product LinePierce™
Quantity30 reactions
TypeChIP Kit
Sufficient For30 Reactions
FormatKit
Unit SizeEach
Contents & Storage
• 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

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