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ChIP-IT® PBMC

可溶化が困難なPBMCのChIPアッセイを簡単に

リンパ球(T細胞, B細胞, NK細胞)や単球などの末梢血単核球(Peripheral Blood Mononuclear Cells ; PBMCs)は, 末梢免疫系の重要な構成要素です。PBMCは免疫学および感染症研究にとって重要であるため, これらの細胞からChIPおよびChIP-Seq解析用の高品質なクロマチンを抽出するための確実な手法が必要とされます。アクティブ・モティフ社は, このような難溶性の試料を用いた実験を何年も重ね, PBMCから高品質なクロマチンを得ることができるように, 最適化されたクロマチン調製法の開発に取り組みました。ChIP-IT® PBMC Kitは, 最適化されたバッファーと溶解条件により, PBMCのように扱いの難しい細胞を用いる場合にも, ChIP反応を成功させることができます。

PBMC ChIP on CD8+ T Cells
ChIP-IT PBMC data for H3K9ac.

ChIP-IT® PBMC の詳細は, 下記の Methodタブ, Dataタブ, もしくは Contentsタブをクリックしてください。マニュアルやカタログ等は, 下記の Documentsタブをクリックしてください。

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.

 
Name Format Cat No. 価格 (税抜)  
ChIP-IT® PBMC 16 rxns 53042 ¥89,000 Buy Now
ChIP-IT® qPCR Analysis Kit 10 rxns 53029 ¥21,000 Buy Now
High Sensitivity Chromatin Preparation 16 rxns 53046 ¥47,000 Buy Now

How does ChIP-IT® PBMC work?

Diagram demonstrating the method of the ChIP-IT High Sensitivity Kit from Active Motif
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.

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.

ChIP-IT PBMC data using chromatin extracted from CD8+ selected T cells showing H3K9ac enrichment at specific loci
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.

qPCR analysis showing H3K4me3 enrichment in three PBMC samples tested in triplicate
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.

ChIP-Seq data of chromatin from T cells for OGT/O-GlcNAc transferase antibody
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.

ChIP-Seq data for Histone H3K4me2 antibody using chromatin from mouse primary T cells
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 & 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