ChIP-IT® PBMC
ChIP-IT-PBMC-AM975
Synonyms
| Name | Format | Cat No. | Price |
|---|
Overview
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.
What our customers are saying about us...
"The ChIP-IT PBMC kit works perfectly for chromatin analysis of primary CD14+ monocytes. It is simple to use, with a straight forward protocol and produces incredibly reproducible data with our cell models of cytomegalovirus infection. In our hands, the ChIP-validated antibodies also prove very reliable."
Ian Groves, PhD
Associate Principal Investigator
Department of Medicine
University of Cambridge
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.
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.
Method
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.
Data
ChIP-IT® PBMC
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.
PBMC chromatin was prepared according to the manual using 20 million PBMCs per reaction. ChIP was performed using the Histone H3K4me3 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. 71002) showed no enrichment, while the positive control primer sets showed nice enrichment of H3K4me3 in the three PBMC samples 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.
ChIP-Seq Validation of ChIP-IT PBMC enriched DNA
The ChIP'd DNA obtained from the ChIP-IT PBMC Kit is of high quality and can be used in downstream applications such as ChIP-Seq or ChIP-chip. Active Motif has validated ChIP-IT PBMC for ChIP-Seq as shown below.
Figure 3: ChIP-Seq data for OGT/O-GlcNAc transferase antibody using chromatin from primary mouse T cells.
ChIP was performed using the ChIP-IT PBMC Kit, 5 µl of OGT/O-GlcNAc transferase antibody (Catalog No. 61355) and chromatin from primary mouse T cells. ChIP DNA was sequenced using the Illumina® Next-Gen sequencing platform and 14 million sequence tags were mapped to identify OGT binding sites. The image on the left shows many OGT binding sites within a 25 million bp region on mouse chromosome 8. The image on the right shows that binding sites are localized to gene promoters as expected based on the known interaction with the H3K4 methylating COMPASS complex.
Figure 4: ChIP-Seq data for Histone H3K4me2 antibody using chromatin from mouse primary T cells.
ChIP was performed using the ChIP-IT PBMC Kit, 5 µl of H3K4me2 antibody (Catalog No. 39141) and chromatin from primary mouse T cells. ChIP DNA was sequenced using the Illumina® Next-Gen sequencing platform and 25 million sequence tags were mapped to identify H3K4me2 binding sites. The image shows the expected H3K4me2 binding pattern with enrichment at promoters and at locations outside of promoters.
Contents
Contents & Storage
The ChIP-IT® PBMC Kit contains sufficient reagents to perform 16 chromatin preparation and immunoprecipitation reactions. Reagents for DNA purification are also provided.
The ChIP-IT PBMC Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not re-freeze the Protein G Agarose Beads after you have received this kit. This kit includes the following components:
- RNase A (10 mg/ml); Store at -20°C
- Proteinase K (10 mg/ml); Store at -20°C
- Blocker; Store at -20°C
- 100 mM PMSF; Store at -20°C
- Protease Inhibitor Cocktail; Store at -20°C
- Precipitation Buffer; Store at -20°C
- Carrier; Store at -20°C
- 10X PBS; Store at -20°C
- Protein G Agarose beads; Store at 4°C
- Swelling Buffer; Store at 4°C
- TE, pH 8.0; Store at RT
- Detergent; Store at RT
- Stop Solution; Store at RT
- 5 M NaCl; Store at RT
- ChIP Filtration Columns; Store at RT
- ChIP Buffer; Store at RT
- Wash Buffer AM1; Store at RT
- Elution Buffer AM4; Store at RT
- DNA Purification Binding Buffer; Store at RT
- 3 M Sodium Acetate; Store at RT
- DNA Purification Wash Buffer; Store at RT
- DNA Purification Elution Buffer; Store at RT
- DNA Purification Columns; Store at RT
Please note that the High Sensitivity Chromatin Preparation Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not re-freeze the Protein G Agarose Beads after you have received this kit. This kit includes the following components:
- RNase A (10 µg/µl); Store at -20°C
- Proteinase K (10 µg/µl); Store at -20°C
- 100 mM PMSF; Store at -20°C
- Protease Inhibitor Cocktail (PIC); Store at -20°C
- Precipitation Buffer; Store at -20°C
- Carrier; Store at -20°C
- 10X PBS; Store at -20°C
- Fixation Buffer; Store at 4°C
- Swelling Buffer; Store at 4°C
- Stop Solution; Store at RT
- Chromatin Prep Buffer; Store at RT
- ChIP Buffer; Store at RT
- DNA Purification Elution Buffer; Store at RT
- 5 M NaCl; Store at RT
- TE, pH 8.0; Store at RT
- Detergent; Store at RT