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Nucleosomes: The Next Generation of Cancer Biomarkers
A nucleosome, composed of histone proteins and the DNA wrapped around them, is the core unit of chromatin. The affect nucleosomes have on chromatin structure and gene regulation is dependent upon the identity of the histones that make up the nucleosome, as well as any modifications to these histone proteins. With a plethora of histone protein variants and post-translational modifications that can occur on their "histone tails", there are a vast number of possibilities when it comes to the exact identiity of a nucleosome and its function. Recently, some of these variants and modifications have been found to associate with cancers. Furthermore, just the total amount of nucleosomes detected can also indicate a disease state or correlate with efficacy of therapy.
Recently, "liquid biopsies", or blood draws from which you can examine biomarkers, have taken hold as an easy, less invasive method to monitor disease state and treatment efficacy. Nucleosomes, histones, and DNA can be shed into the blood during cancer due to high cellular turnover or in response to chemotherapy. Free-floating DNA (cfDNA and ctDNA) is an attractive biomarker, but has shown to be less stable than nucleosomes. Additionally, next generation sequencing of DNA is cumbersome and costly compared to a high-throughput ELISA assay to detect intact nucleosomes. Nucleosome-specific detection assays represent a new generation of biomarker tests: easy, fast, cost-effective, and compatible with stable proteins in liquid biopsy samples.
Histone H3
Active Motif’s Nu.Q® H3 Assay Kit is designed for the detection of levels of H3-containing, cell-free circulating nucleosomes (cf-nucleosomes) in human serum in a high-throughput format. This allows examination of intact H3-containing nucleosomes, regardless of protein variant or any histone modifications. To specifically examine H3.1 (the canonical H3 variant), use our Nu.Q® H3.1 Assay. For added convenience and a more quantitative interpretation of results, the Nu.Q® H3 Assay Kit also include a recombinant nucleosome protein for use as a reference standard curve. For complete details, click the Nu.Q® Method tab below.
Why use Nu.Q® H3?
- Sensitivity: Detect circulating nucleosomes in as little as 10 µl of serum
- Specificity: Nucleosome epitope specific antibody enables detection of only intact nucleosomes
- Quantification: Recombinant nucleosomes enable relative quantification of total levels of circulating nucleosomes
- Convenience: Colorimetric assay in a simple 96-stripwell format for high and low throughput
- Fast: Results can be obtained in 5 hours
Video demonstrating the Nu.Q® assay technology.
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Active Motif's Nu.Q® H3 Assay Kit is powered by VolitionRx. |
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Nu.Q® H3 Assay
Nucleosomes are repeating subunits of DNA and histone proteins that constitute human chromatin. Increased levels of nucleosomes in the blood have been associated with a number of pathological conditions and likely constitutes a major proportion of the cell free DNA (cfDNA) found in the bloodstream. Thus, quantification of the levels of circulating nucleosomes in serum gives insight into the progression of many conditions. The Nu.Q® H3 Assay enables high-throughput screening of cell-free circulating nucleosomes, regardless of histone modification states. This assay is able to detect levels of circulating, H3-containing nucleosomes, from as little as 10 µl of human serum. (Figure 1).
Figure 1: Circulating, H3-containing cell-free nucleosome detection.
The Nu.Q® H3 Assay Kit was used to assay human serum samples. The provided Positive Control was assayed from 0.039 - 2.5 µg/ml as a reference standard curve. Data shown are the results from wells assayed in duplicate with a 20 minute developing time. These results are provided for demonstration only.
The Nu.Q® Assay Method
The Nu.Q® H3 Assay Kit is a sandwich ELISA designed for the detection of levels of H3-containing cell-free circulating nucleosomes (cf-nucleosomes) in human serum. Its high-throughput format enables a large number of samples to be screened simultaneously. The assay utilizes a histone H3 monoclonal antibody to capture nucleosomes from your serum samples. Subsequently a biotinlylated recombinant antibody specific to a common nucleosome epitope is used for detection to assure that only intact nucleosomes are quantified in the assay. Streptavidin conjugated to horseradish peroxidase (SA-HRP) and developing solutions provide a sensitive colorimetric readout that is easily quantified by spectrophotometry. The assay is able to detect levels of circulating H3-containing nucleosomes, regardless of the modification state, from as little as 10 µl of human serum.
Included is a recombinant nucleosome made using Active Motif's patented protein synthesis technology. This protein can be used to build a reference standard curve to determine the total amount of circulating nucleosomes in your samples.
Flow Chart of Nu.Q® Assay Method
Figure 1: Flow chart of the Nu.Q® Assay Method.
The Nu.Q® H3 Assay Method utilizes a histone H3 antibody to capture nucleosomes from samples and a nucleosome epitope specific antibody for detection. Streptavidin conjugated to horseradish peroxidase and developing solutions provide a colorimetric readout that is quantified by spectrophotometry. (Click image to enlarge.)
Contents & Storage
Nu.Q® H3 Assay Kits include a 96-stripwell Total Nucleosome Capture Plate, Total Detection Antibody, Detection Diluent, Streptavidin-HRP Conjugate, Assay Buffer, 10X Wash Buffer, Substrate Solution, Stop Solution, Total Recombinant Nucleosome Positive Control, Control Diluent, Stop Solution and plate sealer. Storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.
