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.
• 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