Active Motif’s ChIP-IT® PBMC Kit overcomes the challenges of generating quality chromatin for use in ChIP from peripheral blood mononuclear cells (PBMCs). PBMCs, including lymphocytes (T cells, B cells and NK cells), monocytes and dendritic cells, constitute a critical component of the peripheral immune system and are often used in immunology, cancer and infectious disease research.
ChIP-IT PBMC data for H3K9ac.
However, for researchers who want to perform ChIP, PBMCs make it difficult to extract quality chromatin because they are highly resistant to lysis under conditions normally suitable for other cell types. Active Motif leveraged its years of experience working with these difficult-to-lyse cells into developing a kit to finally enable researchers to prepare chromatin and perform ChIP on PBMCs. The ChIP-IT PBMC Kit offers a complete solution for chromatin immunoprecipitation including specialized reagents and protocols to enable extraction of high quality chromatin from PBMCs and integrated components and protocols for ChIP and DNA purification from our highly popular ChIP-IT High Sensitivity® Kit.
To learn more about ChIP-IT® PBMC, click on the Method, Data, or Contents tabs below. To view a manual or other related documents, click on the Documents tab below.
ChIP-IT® PBMC Advantages
- Obtain quality chromatin from PBMCs including T & B lymphocytes, dendritic cells and monocytes
- Sensitive enrichment of DNA from nanogram quantities of chromatin
- Optimized reagents from our ChIP-IT High Sensitivity Kit improve signal and minimize background
- Filtration based washes offer a faster, easier solution with better consistency than magnetic capture
- Highly robust procedure has been validated in both qPCR and ChIP-Seq
Active Motif also offers products compatible with ChIP-IT® PBMC:
In addition, our Epigenetic Services can perform ChIP, ChIP-Seq and many other genome-wide data generation and analysis services for you.
How does ChIP-IT® PBMC work?
Figure 1: Schematic of chromatin immunoprecipitation using ChIP-IT PBMC.
In ChIP-IT® PBMC, intact cells are fixed with formaldehyde, which cross-links and preserves protein/DNA interactions. DNA is then sheared into small fragments using sonication and incubated with an antibody directed against the DNA-binding protein of interest. The antibody-bound protein/DNA complexes are immunoprecipitated through the use of Protein G agarose beads and washed via gravity filtration. Following immunoprecipitation, the DNA cross-links are reversed, the proteins are removed by Proteinase K and the DNA is recovered and purified. ChIP enriched DNA can be used for either locus-specific or whole-genome analysis.
The ChIP-IT® PBMC Kit is specially formulated to perform ChIP from PBMCs, including lymphocytes (T cells, B cells and NK cells), monocytes and dendritic cells. ChIP-IT PBMC offers a complete solution for chromatin immunoprecipitation including specialized reagents and protocols to enable extraction of high quality chromatin from PBMCs and integrated components and protocols for ChIP and DNA purification from our highly popular ChIP-IT High Sensitivity® Kit to reduce background and minimize sample loss for improved sensitivity and consistency.
Figure 1: ChIP-IT PBMC data using chromatin extracted from CD8+ selected T cells showing H3K9ac enrichment at specific loci.
T cell chromatin was prepared according to the manual using 20 million CD8+ sorted T cells per reaction. ChIP was performed using Histone H3K9ac antibody on 10 µg of chromatin. Following enrichment, qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029) to normalize the data. The negative control primer set (Catalog No. 71001) showed no enrichment, while both the ACTB-2 (Catalog No. 71005) and GAPDH-2 (Catalog No. 71006) positive control primer sets showed nice enrichment of H3K9ac in the two T cell populations tested. The data represents triplicate values expressed as Binding events detected per 1,000 cells. To convert this scale to the percent of ChIP input recovered, divide the values by 1,000.
Figure 2: ChIP-IT PBMC utilizes the immunoprecipitation protocol from the ChIP-IT High Sensitivity Kit for improved sensitivity and reproducibility.