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ChIP-IT® High Sensitivity

転写因子抗体や幹細胞試料を用いたChIP解析が可能

アクティブ・モティフ社では,存在量の少 ない転写因子,力価の低い抗体,限られた 量の試料を扱う研究者のニーズに応えるべく,ChIP-IT® 製品をさらに拡充しました。ChIP-IT High Sensitivity® Kitは,DNA-タンパク質間相互作用や抗体結合力が低い場合においても,ChIP-Seq,ChIP-chip,qPCRなどの下流処理で確実に検出できるように,シグナル強度を高め,バックグラウンドを低くしています。

ChIP-IT High Sensitivity® Kitでは,培養細胞,新鮮組織,凍結組織からクロマチンを調製するために,特別に用意されたバッファーを使用します。免疫沈降反応中の非特異的結合を最小限に抑えるために,低バックグラウンドのプロテインGアガロース ビーズと抗体ブロッキング剤を使用し,市販されている他のChIP Kitよりも高い感度を実現しています(下記Dataタブの図1)。ろ過カラムを用いることにより,洗浄工程を,迅速かつ簡便にし,再現性の高い溶液を得ることが可能になりました。qPCR やChIP-Seqには,質の高いChIP DNAが求められるため,DNA精製試薬もキットに含まれています。

ChIP-IT High Sensitivity® Kitと互換性があるキット:

  • ChIP-IT® qPCR Analysis Kit – qPCR データ解析を簡素化し、複数の試料間および実験間で比較できるようデータを標準化することができるキットです。
  • qPCR Primer Sets – モデル生物を用いたChIP-qPCR用コントロールプライマーセットを取扱っております。
  • Antibodies – ChIP-IT High Sensitivity Kit と共に使用して高品質なChIPデータを得ることができる抗体を取扱っております。

さらに, 試料調製、ChIPアッセイからデータ解析までを全て、熟練されたアメリカ本社の研究者が行うChIPアッセイ受託サービスを承っております。ChIP-Seq や ChIP-qPCRを行い、高品質なデータを直接納品致します。なお、お手持ちの抗体がChIP-Seqに適しているか解析するサービスも実施中です。抗体のChIP-Seq適性評価受託サービスの詳細はこちらからご覧ください。

 
Name Format Cat No. 価格 (税抜)  
ChIP-IT High Sensitivity® 16 rxns 53040 ¥93,000 Buy Now
High Sensitivity Chromatin Preparation 16 rxns 53046 ¥47,000 Buy Now
ChIP-IT® Fixation Buffer 3 ml 53038 ¥18,000 Buy Now
Protein G Agarose Columns 30 rxns 53039 ¥60,000 Buy Now
5 rxns 53037 ¥14,000 Buy Now
Protein G Agarose Beads 1.2 ml 37499 ¥20,000 Buy Now
TE, pH 8.0 35 ml 37515 ¥7,800 Buy Now
ChIP Buffer 50 ml 37516 ¥9,200 Buy Now
Blocker 0.1 ml 37498 ¥13,000 Buy Now

ChIP-IT High Sensitivity® Advantages

  • Ideal for low abundance transcription factors or antibodies with low binding affinities
  • Sensitive enrichment of DNA from as little as 1,000 cells per immunoprecipitation reaction for high abundance target proteins and as little as 50,000 cells for low abundance transcription factors
  • Optimized reagents reduce background levels caused by non-specific binding events
  • Filtration based washes offer a faster, easier solution with better consistency than magnetic capture
  • Highly robust procedure has been validated across multiple sample types with proven performance in both qPCR and ChIP-Seq analysis

How does the ChIP-IT High Sensitivity® Kit work?

Diagram demonstrating the method of the ChIP-IT High Sensitivity Kit from Active Motif
Figure 1: Schematic of chromatin immunoprecipitation using ChIP-IT High Sensitivity.

In ChIP-IT High Sensitivity®, intact cells are fixed with a specially formulated formaldehyde buffer, 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 gene-specific or whole-genome analysis.

Detect Low Abundance Protein Targets

The ChIP-IT High Sensitivity® Kit is ideal for use with challenging antibodies that do not give signal with other ChIP methods, as the method is sensitive enough to detect specific binding of even low abundance proteins. Below is a comparison of ChIP-IT High Sensitivity with other commercially available ChIP kits. ChIP was performed using untreated MCF-7 chromatin and an antibody that binds the low abundance nuclear receptor co-activator 2 (NCOA2) transcriptional co-factor. It was also performed with a negative control IgG and a negative control primer set (Active Motif Catalog No. 71001) to monitor non-specific binding. Only the ChIP-IT High Sensitivity method was able to detect NCOA2 binding at specific genomic locations (ESRRA promoter), providing approximately 20-fold higher enrichment than the negative controls. Although ESRRA shows qPCR signals using the kit from Competitor D, the enrichment levels are poor due to the high background from the negative control primer set and the non-specific binding detected with the IgG.

PCR analysis showing specific enrichment from low abundance target proteins
Figure 1: Comparison of ChIP kits targeting a low abundance transcription factor.

MCF-7 chromatin was prepared according to each manufacturer's recommendations for their ChIP assay from 1.5 x 106 cells. ChIP was performed with the optimal amount of chromatin as suggested by the protocol for each manufacturer using an antibody for the low abundance NCOA2 protein and a negative control IgG. Following enrichment, qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029) in order to normalize the data and allow for direct comparison of the results. Data is expressed as binding events detected per 1,000 cells which represents the average of the raw data triplicates adjusted for the amount of chromatin in the reaction, the resuspension volume and the primer efficiency.


Achieve Greater Sensitivity

A comparison of ChIP-IT High Sensitivity to other commercially available ChIP Kits when using the high-quality H3K4me3 antibody (Catalog No. 39915) shows specific signal for the positive control GAPDH promoter (Catalog No. 71006) using ChIP-IT HS, Competitor M and Competitor I's Kits. Due to the optimized conditions with the ChIP-IT High Sensitivity, the detectable enrichment was 2-3 fold higher than the enrichment obtained from Competitor M and Competitor I. Only Competitor D did not show good enrichments as a result of high non-specific binding as seen with the signal from the negative control qPCR primer set and the high signal in the IgG reactions.

PCR analysis showing specific enrichment from low abundance target proteins
Figure 2: ChIP-IT High Sensitivity shows better enrichment than competitor ChIP Kits.

MCF-7 chromatin was prepared according to each manufacturer's recommendations for their ChIP assay from 1.5 x 106 cells. ChIP was performed with the optimal amount of chromatin as suggested by the protocol for each manufacturer using an antibody for the abundant Histone H3K4me3 (Catalog No. 39915) and a negative control IgG. Following enrichment, qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029) in order to normalize the data and allow for direct comparison of the results. Data is expressed as binding events detected per 1,000 cells which represents the average of the raw data triplicates adjusted for the amount of chromatin in the reaction, the resuspension volume and the primer efficiency.


Use Less Sample Material

The optimized ChIP buffers included in the ChIP-IT High Sensitivity Kit reduce the presence of non-specific DNA, thereby minimizing background levels. This results in a increased sensitivity and better enrichment. The reduced background makes it possible to use the ChIP-IT High Sensitivity Kit with as little as 1,000 cells per immunoprecipitation reaction for high abundance proteins and as little as 50,000 cells per immunoprecipitation reaction for low abundance proteins. In the experiment below, ChIP was performed using the Histone H3K4me3 antibody and chromatin from 1,000 to 1 million cells. Significant enrichment was still detectable at the positive control GAPDH promoter when using only 1,000 cells.

PCR analysis showing successful ChIP using samples from 1,000 cells
Figure 3: ChIP-IT High Sensitivity is suitable for use with limited sample material.

MCF-7 chromatin was prepared according to the ChIP-IT High Sensitivity manual using between 100,000 to 4 million cells per chromatin preparation. The immunoprecipitation was then performed using the indicated number of cells with Active Motif's Histone H3K4me3 antibody (Catalog No. 39915) and a negative control IgG. Following enrichment, qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029). The ChIp-IT High Sensitivity Kit shows strong enrichment with the positive control GAPDH promoter from as little as 1,000 cells and no enrichment with the negative control Untr12 primer set. Data is expressed as binding events detected per 1,000 cells which represents the average of the raw data triplicates adjusted for the amount of chromatin in the reaction, the resuspension volume and the primer efficiency.


Enriched DNA is ChIP-Seq Validated

The ChIP DNA obtained from the ChIP-IT High Sensitivity Kit is of high quality and can be used in downstream applications such as ChIP-Seq or ChIP-chip. Active Motif has validated the ChIP-IT High Sensitivity Kit for ChIP-Seq with both challenging antibodies that target low abundance proteins and highly abundant histone proteins as shown below.

ChIP-Seq data for the low abundance target NCOA
Figure 4: ChIP-Seq data for the low abundance transcription factor NCOA2.

ChIP was performed using the ChIP-IT High Sensitivity Kit, an antibody against the low abundance transcriptional co-activator NCOA2 and chromatin from 3 million MCF-7 cells. ChIP DNA was processed and 30 million sequence tags were generated using the Illumina® Next-Gen sequencing platform. The sequence data was analyzed to identify all NCOA2 binding sites in the human genome. The left panel shows multiple binding sites across a 1 million base pair region on chromosome 11. The image in the right panel is zoomed in to show NCOA2 peaks in the promoters of two genes.

ChIP-Seq data using Histone H3K4me3
Figure 5: ChIP-Seq data for Histone H3K4me3.

ChIP was performed using the ChIP-IT High Sensitivity Kit, an antibody against Histone H3K4me3 (Catalog No. 39915) and chromatin from 1 million MCF-7 cells. ChIP data was processed and 24 million sequence tags were generated using the Illumina® Next-Gen sequencing platform. The sequencing data was analyzed to identify all the H3K4me3 binding sites in the human genome. The left panel shows the presence of the H3K4me3 in the promoters of 5 genes across a 175,000 bp region on chromosome 11. The image in the right panel is zoomed in to show binding in the promoter of a single gene. Binding at the PPFIA1 gene shows the common double peak with depletion right at the transcriptional start site.

Contents & Storage

Please note that the ChIP-IT High Sensitivity 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
  • Blocker; Store at -20°C
  • Fixation Buffer; Store at 4°C
  • Protein G Agarose beads; Store at 4°C
  • Stop Solution; Store at RT
  • Chromatin Prep Buffer; Store at RT
  • ChIP Buffer; Store at RT
  • Wash Buffer AM1; Store at RT
  • Elution Buffer AM4; Store at RT
  • ChIP Filtration Columns; Store at RT
  • 5 M NaCl; Store at RT
  • TE, pH 8.0; Store at RT
  • Detergent; 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