|To date, the most widely used and powerful method to identify regions of the genome associated with specific proteins is the Chromatin Immunoprecipitation (ChIP) assay. The ChIP assay has been widely used to study both histones and non-histone proteins, such as transcription factors, within the context of the cell. Transcription factors and other DNA-binding proteins have a weaker affinity than histones, which generally are tightly associated within the chromatin complex. To avoid dissociation of non-histone proteins from the chromatin binding site, it is necessary to incorporate a cross-linking step. Importantly, antibodies that recognize a protein of interest can be used to determine the relative association of that antigen in the context of chromatin at one or more loci in the genome.|
MAGnify™ Chromatin Immunoprecipitation System
This kit provides a faster and more reproducible solution for Chromatin Immunoprecipitation (ChIP) and includes all reagents needed to perform ChIP with an antibody of interest. With the MAGnify™ Chromatin Immunoprecipitation System you will:
- Reduce overall ChIP protocol time by one day (Table 1)
- Reduce input cell number per ChIP experiment (10K–300K cells required) (Figures 1,2)
- Decrease background caused by nonspecific binding through the use of Dynabeads®
- Improve reproducibility due to optimized magnetic DNA purification (avoid columns and phenol/chloroform steps) (Figure 3)
- Increase confidence in results due to optimized and reproducible components and antibodies qualified in Chromatin Immunoprecipitation
- Easily increase throughput with small volumes, magnetic protocol, and magnet compatible with multi-channel pipetting
- Reduce experimental error with Dynabeads® Protein A/G Mix—worry less about antibody compatibility
The ChIP-ready DNA is ready for downstream analysis for most methods, including PCR/qPCR-based assays, bisuflite conversion followed by amplification, cloning, sequencing, direct sequencing, library preparation for high-throughput sequencing, and sample-prep for DNA microarray analysis. This kit is unique in its utility for permitting researchers to take advantage of lower starting cell numbers for ChIP, thus preserving precious samples such as primary cells, stem cells, and biopsies. A key aspect for a successful ChIP is having antibodies that recognize the target protein in the context of chromatin.
Table 1. Comparison to a conventional ChIP protocol.
|Workflow step||MAGnify™ ChIP timeline||Conventional ChIP timeline|
|Antibody/chromatin incubation||2 hr||Overnight|
|Bead pulldown||1 hr||2 hr|
|Washes||30 min (2 buffers)||1- 3 hr (4 buffers)|
|Reverse crosslinking||1.5 hr||Overnight|
|Proteinase K digestion||2 hr|
|DNA elution from beads||15-30 min|
|DNA purification||2 hr- overnight|
|Average time||5 hours||36-48 hours|
Figure 1. Titration of cell number with MAGnify™ ChIP.
Sheared chromatin from MCF7 cells was prepared from 1 million cells in 50 μl lysis buffer and diluted to indicated number of cells per ChIP according to the MAGnify™ ChIP protocol. 1 μg of antibody (IgG–included in kit, H3-K27Me3–Cat. no. 49-1014, H3-K9Ac–Cat. no. 49-1009) were used for each ChIP experiment. 1% of the sonicated chromatin was set aside as input control. Optimized qPCR primers were used to amplify the SAT2 locus (Cat. no. 49-2026). Data are graphed as percent input.
Figure 2. 10,000 cell input with MAGnify™ ChIP.
Sheared chromatin from MCF7 or MDA-MB231 cells was prepared from 1 million cells in 50 μl lysis buffer and diluted to 10,000 cells per ChIP, according to the MAGnify™ ChIP protocol. 1 μg of antibody (IgG—included in kit, H3-K27Me3—Cat. no. 49-1014, H3-K9Ac—Cat. no. 49-1009) or 3μl RNA pol II (Cat. no. 49-1033) were used for each ChIP experiment. 1% of the sonicated chromatin was set aside as input control. Optimized qPCR primers were used to amplify to different loci: RARb1 promoter (Cat. no. 49-2027) or ER-a promoter (Cat. no. 49-2028). Data are graphed as percent input.
Figure 3. MAGnify™ ChIP versus conventional protocols.
Sheared chromatin from 293Gt cells was prepared and 50,000 cells used for ChIP (MAGnify™ ChIP) and 1 million cells per ChIP used for the conventional ChIP protocol. 1 μg of antibody (IgG— included in kit, H3-K9Me3—Cat. no. 49-1008) was used for each ChIP experiment. 1% of the sonicated chromatin was set aside as input control. Optimized qPCR primers were used to amplify the SAT2 locus (Cat. no. 49-2026), a known target of heterochromatin-associated H3-K9Me3. Key elements of a conventional protocol use an overnight immunoprecipitation step of antibody and chromatin, followed by enrichment with Protein A sepharose beads and extensive washes with buffers containing variable salt concentrations. Reverse crosslinking was done overnight at 65°C and DNA purification performed by phenol-chloroform extraction.