由147个碱基对条形码widom601定位序列DNA缠绕在由大肠杆菌表达的重组人组蛋白组装而成的一组明显修饰的单核小体(组蛋白H2A、H2B、H3和H4各2个;accession numbers:H2A-P04908、H2B-O60814、H3.1-P68431或H3.2-Q71DI3*、H4-P62805)，由1个未修饰的加上15个组蛋白H3或H4翻译后修饰（PTMs，通过专有的半合成方法创建）组成：H3K4、K9、K27和H4K20与me1、me2或me3。每个明显修饰的核小体都可以通过3'端的独特DNA序列（“barcode”）进行区分，该序列可以通过qPCR或二代测序进行破译。池中的16个核小体都被2种不同物种的DNA缠绕，每一种都含有独特的条形码(“A”和“B”，参考SNAP-ChIP手册)。适合用作ChIP、抗体特异性测试或效应蛋白结合实验的掺入质控品。

*组蛋白H3.2在110位包含一个Cys到Ala的替换。

产品详情

保存温度: Stable for six months at -20°C from date of receipt.

运输温度: DO NOT FREEZE!! Frozen cold packs.

产品形式: Purified recombinant mononucleosomes, containing a mixture of 16 (1 unmodified plus 15 unique) H3 and H4 PTMs in 10 mM sodium cacodylate pH 7.5, 100 mM NaCl, 1 mM EDTA, 50% glycerol (w/v), 1x Protease Inhibitor cocktail, 100 µg/mL BSA, 10 mM β-mercaptoethanol. Average molarity = 0.6 nM. MW = ~199382.1 Da (average MW of all 16 nucleosomes).

验证数据

Fig 1. DNA Gel Data: Representative images for SNAP-ChIP K-MetStats (H3K4me0 = unmodified, H3K4me2, H3K4me3) run on a native PAGE gel and stained with ethidium bromide to visualize DNA. Lane 1: Free 147bp DNA used in nuclesome assembly (100 ng). Lane 2: Intact nucleosomes (200 ng) showing lack of free DNA. Identical experiments were performed for the entire K-MetStat Panel.

Fig 2. Protein Gel Data: Representative Coomassie stained PAGE gel for SNAP-ChIP K-MetStats (2 µg each of unmodified, H3K4me1, H3K4me2, H3K4me3) to demonstrate the purity of the histones in the preparation. Sizes of molecular weight markers and positions of the core histones (H2A, H2B, H3 and H4) are indicated. Identical experiments were performed for the remainder of the K-MetStat Panel.

Fig 3. ChIP Data: Representative images for SNAP-ChIP K-MetStats (unmodified, H3K4me1, H3K4me2, H3K4me3) assayed in a chromatin immunoprecipitation (ChIP) experiment using commercially available ChIP grade antibodies (3 µg, n = 3). Quantitative Real-Time PCR (qPCR) for the DNA barcodes corresponding to unmodified (H3K4me0), H3K4me1, H3K4me2, and H3K4me3 nucleosomes show recovery of the barcodes corresponding to the expected antibody target. Identical experiments were performed for the remainder of the K-MetStat Panel (H3K9me1, H3K9me2, H3K9me3, H3K27me1, H3K27me2, H3K27me3, H3K36me1, H3K36me2, H3K36me3, H4K20me1, H4K20me2 and H4K20me3).

Fig 4. ChIP Data: Representative chromatin immunoprecipitation (ChIP) data using commercially available ChIP-grade antibodies targeting each PTM  in the K-MetStat panel.  The antibodies were assayed in a native ChIP experiment with 3 μg antibody added to 3 μg K-562 cell chromatin with  the K-MetStat Panel spiked-in prior to micrococcal nuclease digestion.  Quantitative real-time PCR (qPCR) was used to measure recovery of  duplicate DNA barcodes corresponding to the indicated panel nucleosomes (blue bars, x-axis).  The black bars map to the log scale on the right  y-axis and indicate the percentage of target immunoprecipitated relative to the input (a measure of the antibody efficiency).  In each case, the SNAP-ChIP spike-in confirmed that the antibodies recovered the expected histone PTM.  Enrichment of off-target PTMs is due to antibody  cross-reactivity.

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