ChIP-IT® High Sensitivity
ChIP for limited sample material and low abundance target proteins
The ChIP-IT® High Sensitivity (HS) Kit is designed to give superior performance when performing ChIP using antibodies against low abundance transcription factors, antibodies with sub-optimal binding affinities or when starting with a limited number of cells. Higher signal and lower non-specific DNA pulldown is achieved through the incorporation of specialized protein G agarose beads and optimized fixation, binding and wash buffers. In addition, washes are performed via gravity filtration, which is faster, easier and more consistent than magnetic bead capture. The high-quality ChIP DNA obtained from the ChIP-IT High Sensitivity Kit is also ideal for ChIP-Seq; ChIP-IT HS has been validated for use in this application, even when using antibodies that target low abundance transcription factors.
Active Motif also offers products compatible with the ChIP-IT High Sensitivity Kit:
- ChIP-IT® qPCR Analysis Kit – simplifies qPCR data analysis and enables normalization across multiple sample types and experiments
- qPCR Primer Sets for a variety of popular gene targets in a number of model organisms.
- Antibodies validated with our ChIP-IT High Sensitivity Kit.
In addition, our Epigenetic Services can perform ChIP, ChIP-Seq and many other genome-wide data generation and analysis services for you.
|ChIP-IT® High Sensitivity||16 rxns||53040||$440||Buy Now|
|Protein G Agarose Columns||30 rxns||53039||$300||Buy Now|
|ChIP-IT® Fixation Buffer||3 ml||53038||$75||Buy Now|
|ChIP-IT® Fixation Buffer|
|Protein G Agarose Columns Data Sheet|
|ChIP-IT® High Sensitivity Kit Manual|
|Chromatin & Epigenetics Products Brochure|
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?
Figure 1: Schematic of chromatin immunoprecipitation using ChIP-IT High Sensitivity.
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.
Figure 1: Comparison of ChIP kits targeting a low abundance transcription factor.
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.
Figure 2: ChIP-IT High Sensitivity shows better enrichment than competitor ChIP Kits.
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.
Figure 3: ChIP-IT High Sensitivity is suitable for use with limited sample material.
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.
Figure 4: ChIP-Seq data for the low abundance transcription factor NCOA2.
Figure 5: ChIP-Seq data for Histone H3K4me3.
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. 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
- Blocker; Store at -20°C
- 5 M NaCl; Store at RT
- 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
- TE, pH 8.0; Store at RT
- Detergent; Store at RT
- 10X PBS; Store at -20°C
- Fixation Buffer; Store at 4°C
- Stop Solution; Store at RT
- Chromatin Prep Buffer; 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
- Protein G Agarose beads; Store at 4°C
- 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