由147個堿基對條形碼widom601定位序列DNA纏繞在由大腸桿菌表達(dá)的重組人組蛋白組裝而成的一組明顯修飾的單核小體(組蛋白H2A、H2B、H3和H4各2個;accession numbers:H2A-P04908、H2B-O60814、H3.1-P68431或H3.2-Q71DI3*、H4-P62805)，由1個未修飾的加上15個組蛋白H3或H4翻譯后修飾（PTMs，通過專有的半合成方法創(chuàng)建）組成：H3K4、K9、K27和H4K20與me1、me2或me3。每個明顯修飾的核小體都可以通過3'端的獨(dú)-特DNA序列（“barcode"）進(jìn)行區(qū)分，該序列可以通過qPCR或二代測序進(jìn)行破譯。池中的16個核小體都被2種不同物種的DNA纏繞，每一種都含有獨(dú)-特的條形碼(“A"和“B"，參考SNAP-ChIP手冊)。適合用作ChIP、抗體特異性測試或效應(yīng)蛋白結(jié)合實(shí)驗(yàn)的摻入質(zhì)控品。

*組蛋白H3.2在110位包含一個Cys到Ala的替換。

產(chǎn)品詳情

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

運(yùn)輸溫度: DO NOT FREEZE!! Frozen cold packs.

產(chǎn)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).

驗(yàn)證數(shù)據(jù)

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.

訂購詳情

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