EpiCypher—优质核小体的选择
在科研实验中，如果您要使用重组核小体进行染色质实验，则需考虑以下几点：
1、用于开发修饰组蛋白的方法。确保使用无疤痕方法整合所需的PTMs是很重要的，这种方法可以重现天然组蛋白结构。EpiCypher 使用几种不同的方法获得修饰的组蛋白，所有方法都会无疤痕整合组蛋白修饰。
为什么这很重要呢？许多市售的重组核小体是使用组蛋白PTM类似物构建的，如甲基赖氨酸类似物(MLAs) 10，其会导致修饰位点的氨基酸序列发生变化。这些非天然组蛋白修饰类似物已被证明会破坏与染色质调节蛋白和组蛋白PTM特异性抗体的相互作用，并且这对研究生理机制来讲是不理想的。因此，使用这些方法合成的核小体时应格外谨慎11-13。
2、修饰组蛋白的纯度。组装完全定义的同质核小体的下一步是对修饰组蛋白进行严格的质量控制。
在质控结果中，HPLC 迹线应显示是单一洗脱物质，表明组蛋白纯度>95%；平行高分辨率质谱（HRMS）应在预期质量的1道尔顿范围内显示一个单峰，没有任何意义的额外电荷质量（m/z）信号（例如图2A）。EpiCypher的所有修饰组蛋白均通过HPLC和HRMS分析进行验证。
为什么这很重要呢？不必要的物质（如甲硫氨酸氧化）可能引起结构改变，并影响静电相互作用或疏水相互作用，从而损害下游核小体组装的效率。
3、组装核小体的质量控制(QC)指标。DNA组装后核小体的质量验证对最终产品的信任保证至关重要。
EpiCypher使用天然PAGE分析DNA上的dNuc组装，其中使用约150bp DNA的高效组装应该只产生单一物质，这相对于未组装的游离DNA，其迁移率降低(图2B，下图)。
为什么这很重要呢？因为被污染的游离DNA会诱导染色质修饰酶(如NSD2)的异常活性，所以必须避免。此外，次优组装可能导致样品的异质性混合，包括错误定位的核小体。
我们还使用考马斯染色对最终的dNucs进行SDS-PAGE分析，以确保四种组蛋白的化学计量相等（图2C，下图）。然后，我们通过免疫印迹证实了整合组蛋白PTM的存在（图2C，上图）。
为什么这很重要呢？如果其他种类的蛋白质污染或偏离1:1:1:1的比例，则可能表明组蛋白降解或组装不良，因此有必要重新解析每个组蛋白。而免疫印迹对于确定修饰是否存在于组蛋白的正确位置上非常重要。
核小体相关产品
分类 | 货号 | 产品名称 |
rNucs Human Recombinant Nucleosomes, No PTMs | 16-0006 | Mononucleosomes, biotinylated |
16-0009 | Mononucleosomes, non-biotinylated | |
16-0024 | Mononucleosomes, desthiobiotinylated | |
16-0027 | Tailless Nucleosomes, biotinylated | |
16-0023 | Mononucleosomes, H3.1 ΔN2, biotinylated | |
16-0016 | Mononucleosomes, H3.1 ΔN32, biotinylated | |
16-1016 | Mononucleosomes, H3.1 ΔN32, non-biotinylated | |
16-0017 | Mononucleosomes, H3.3 ΔN32, biotinylated | |
16-1017 | Mononucleosomes, H3.3 ΔN32, non-biotinylated | |
16-0018 | Mononucleosomes, H4 ΔN15, biotinylated | |
16-3004 | Dinucleosomes, biotinylated | |
16-3104 | Dinucleosomes, non-biotinylated | |
dNucs Designer Recombinant Nucleosomes with PTMs | 16-0321 | H3K4me1, biotinylated |
16-0334 | H3K4me2, biotinylated | |
16-1334 | H3K4me2, non-biotinylated | |
16-0316 | H3K4me3, biotinylated | |
16-1316 | H3K4me3, non-biotinylated | |
16-0402 | H3K4,K9me3, biotinylated | |
16-0403 | H3K4,K27me3, biotinylated | |
16-0335 | H3K4me3,K9,14,18ac, biotinylated | |
16-0325 | H3K9me1, biotinylated | |
16-0324 | H3K9me2, biotinylated | |
16-0315 | H3K9me3, biotinylated | |
16-0338 | H3K27me1, biotinylated | |
16-0339 | H3K27me2, biotinylated | |
16-0317 | H3K27me3, biotinylated | |
16-1317 | H3K27me3, non-biotinylated | |
16-0397 | H3.1K27me3,S28phos, biotinylated | |
16-0322 | H3K36me1, biotinylated | |
16-0319 | H3K36me2, biotinylated | |
16-0320 | H3K36me3, biotinylated | |
16-1320 | H3K36me3, non-biotinylated | |
16-0390 | H3.3K36me3, biotinylated | |
16-0367 | H3K79me1, biotinylated | |
16-0368 | H3K79me2, biotinylated | |
16-0369 | H3K79me3, biotinylated | |
16-0393 | H4K12me1, biotinylated | |
16-0331 | H4K20me1, biotinylated | |
16-0332 | H4K20me2, biotinylated | |
16-0333 | H4K20me3, biotinylated | |
16-1333 | H4K20me3, non-biotinylated | |
vNucs Histone Variants | 16-0013 | H2AX, biotinylated |
16-1013 | H2AX, non-biotinylated | |
16-0366 | H2AXS139phos, biotinylated | |
16-0014 | H2AZ.1, biotinylated | |
16-1014 | H2AZ.1, non-biotinylated | |
16-0015 | H2AZ.2, biotinylated | |
16-0011 | H3.3, biotinylated | |
16-0012 | H3.3, non-biotinylated | |
Mutant Nucs Defined Amino Acid Substitutions | 16-0029 | H2AE61A, biotinylated |
16-1029 | H2AE61A, non-biotinylated | |
16-0030 | H2AE92K, biotinylated | |
16-1030 | H2AE92K, non-biotinylated | |
16-0031 | H2BE105A,E113A, biotinylated | |
16-1031 | H2BE105A,E113A, non-biotinylated | |
16-0349 | Oncogenic Nucs (oncoNucs) | |
16-0350 | H3.3K9M, biotinylated | |
16-1323 | H3.3K27M, biotinylated | |
16-0323 | H3.3K27M, non-biotinylated | |
16-0346 | H3.3G34R, biotinylated | |
16-0347 | H3.3G34V, biotinylated | |
16-0348 | H3.3G34W, biotinylated | |
16-0344 | H3.3K36M, biotinylated | |
Methyl DNA Nucs Nucleosomes with Methylated DNA | 16-2043 | Mononucleosomes, Recombinant, Hemi-methylated 199x601 DNA, biotinylated |
16-2143 | Mononucleosomes, Recombinant, Hemi-methylated 199x601 DNA, non-biotinylated | |
16-2044 | Mononucleosomes, Recombinant, 199x601 DNA, biotinylated | |
16-2144 | Mononucleosomes, Recombinant, 199x601 DNA, non-biotinylated | |
16-2045 | Mononucleosomes, Recombinant, Symmetrically Methylated 199x601 DNA, biotinylated | |
EpiDyne® Chromatin Remodeling Assay Substrates | 16-4201 | EpiDyne FRET Nucleosome Remodeling Assay Substrate |
16-4101 | EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1 | |
16-4112 | EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1,2, biotinylated | |
16-4113 | EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1,2,3, biotinylated | |
16-4114 | EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1, 50-N-66, biotinylated | |
16-4115 | EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1,2, 50-N-66, biotinylated | |
16-4116 | EpiDyne Nucleosome Remodeling Assay Substrate ST601-GATC1,2,3, 50-N-66, biotinylated | |
SNAP Spike-in Controls | 19-1002 | SNAP-CUTANA™ K-MetStat Panel |
19-1001 | SNAP-ChIP K-MetStat Panel | |
19-2001 | SNAP-ChIP OncoStat Panel | |
19-3001 | SNAP-ChIP K-AcylStat Panel | |
dCypher™ Nucleosome Panels | 16-9001 | dCypher™ Nucleosome Full Panel |
想了解更多关于EpiCypher重组核小体技术和产品的信息吗？请联系欣博盛！
EpiCypher的注册商标和知识产权可见链接：https://www.epicypher.com/intellectual-property/。
本文中的所有其他商标和商品均为其各自公司所有。
本文翻译自链接：https://www.epicypher.com/resources/blog/finding-the-best-substrate-for-studying-histone-modifications/，如与原文有出入的地方，请以英文原文为准。
未经EpiCypher公司事先书面同意，本文件不得部分或全部复制。
关于EpiCypher公司：
EpiCypher是一家成立于2012年的表观遗传学公司。从专有组蛋白肽阵列平台EpiGold™开始，EpiCypher开发了一系列同类产品。同时，EpiCypher是重组核小体制造和开发的全球领导者。利用其独有技术，不断增加产品库中高纯度修饰重组核小体（dNucs™）产品。dNuc™多样性的产品为破译组蛋白编码和加速药物开发提供了强大的工具。
EpiCypher还将dNuc™技术广泛的应用于多种分析测定产品中，包括：SNAP-ChIP® Spike-in Controls（用于抗体分析和ChIP定量）， EpiDyne® 底物（用于染色质重塑和抑制剂筛选及开发），dCyher™测定（用于探究表观遗传蛋白质-组蛋白PTM结合相互作用）。最近，EpiCypher还推出了针对ChIC、CUT&RUN和CUT&Tag的高灵敏度表观基因组图谱CUTANA™分析。
如需了解更多详细信息或相关产品，请联系EpiCypher中国授权代理商-欣博盛生物
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