http://www.abcam.cn/epigenetics/histone-modifications-a-guide

组蛋白修饰是一类调控基因表达的翻译后修饰，其中组蛋白 H3 是修饰最多的组蛋白。​​
组蛋白的翻译后修饰 — 又称为标记 — 可将基因组调节成易于 DNA 转录的常染色体活性区域或者 DNA 更紧密，不易于转录的失活异染色质区域，借此调控基因表达。

组蛋白将 DNA 组装并排列成称为核小体的结构，使其适于存储在细胞核中。每个核小体含有两个亚基，两个亚基均由组蛋白 H2A、H2B、H3 和 H4（称之为核心组蛋白）构成，而连接组蛋白 H1 则作为稳定剂。

组蛋白 H3 是修饰最多的组蛋白。组蛋白 H3 的修饰能预测染色质的类型（异染色质或常染色质）、区分基因组功能元件（启动子、增强子、基因主体）以及检测决定这些元件处于活性状态或是抑制状态。

下载表观遗传学修饰海报

图 1.最常见的组蛋白修饰。查看组蛋白修饰海报了解更多信息。

研究组蛋白 H3 的修饰时，最有用的对照是总组蛋白 H3。

相关产品

组蛋白 H3 抗体
ChIP 试剂盒
染色质和组蛋白提取试剂盒
总组蛋白 H3 定量试剂盒 — 比色法或荧光法
​​组蛋白甲基化​​

根据甲基化位点，H3 和 H4 的赖氨酸甲基化可调节转录激活和抑制，而精氨酸甲基化则促进转录激活。1

赖氨酸可被单甲基化、二甲基化或三甲基化，使得每个甲基化位点都能实现功能上的多样性。例如，K4 的单或三甲基化（H3K4me1 或 H3K4me3）均为激活标记，但 H3K4me1 存在于转录增强子中，而 H3K4me3 存在于基因启动子中。K36 的三甲基化 (H3K36me3) 与基因主体的转录区域相关。

组蛋白 H3 上的 K9 和 K27 的三甲基化（H3K27me3 和 H3K9me3）都是抑制信号，但是 H3K27me3 是控制发育调控因子的临时信号。相反，H3K9me3 则是具有串联重复结构的染色体区域形成永久异染色质的信号。

H3K27me3 主要存在于基因密集区域的启动子中，并与胚胎干细胞中的发育调控因子，包括 Hox 和 Sox 基因密切相关。H3K9me3

则通常存在于缺少基因的区域例如卫星重复、端粒和近着丝粒区。另外它也标记反转录转座子和锌指基因中的某些特定家族 (KRAB-ZFP)。

两种标记都存在于失活的 X 染色体中，其中 H3K27me3 位于基因间区域和沉默的编码区，而 H3K9me3 主要出现在活性基因的编码区中。

相关产品
抗 H3K4me3 和 K4me1 抗体 — ChIP 级
抗 H3K9me3 抗体 — ChIP 级
抗 H3K27me3 抗体 — ChIP 级
抗 H3K36me3 抗体 — ChIP 级
H3K4 甲基化检测与定量试剂盒
H3K9 甲基化检测与定量试剂盒
H3K27 甲基化检测与定量试剂盒
组蛋白乙酰化

组蛋白乙酰化通常与开放染色质结构相关。因此染色质可与转录因子接触，并且能够显著提高基因表达的水平。2

组蛋白乙酰化主要出现在启动子区域。例如，组蛋白 H3 的 K9 和 K27 乙酰化（H3K9ac 和 H3K27ac）通常与活性基因的增强子和启动子有关。在转录基因各处也存在低水平乙酰化，但其功能尚不明朗。

组蛋白乙酰转移酶 (HAT) 和去乙酰化酶 (HDAC) 是在组蛋白尾部进行乙酰化修饰和去修饰的酶。组蛋白 H3 和 H4 中的赖氨酸残基是 HAT 复合物的优选靶点。

相关产品

抗组蛋白 H3K9Ac 抗体 | ChIP 级
抗组蛋白 H3K27Ac 抗体 | ChIP 级
H3K9Ac 定量和检测试剂盒
组蛋白磷酸化

核心组蛋白的磷酸化是细胞分裂中染色体凝缩、转录调控和 DNA 损伤修复的关键中间步骤。3–6与乙酰化和甲基化不同，组蛋白磷酸化似乎需要通过与其他组蛋白修饰的相互作用并作为效应子蛋白的平台，才可发挥其功能。这样会引发下游级联事件。

组蛋白 H3 的 S10 磷酸化（S10 磷酸化 H3）和组蛋白 2A 的 T120 的磷酸化是有丝分裂的标志：这些修饰参与有丝分裂期间染色质的收缩以及染色质结构和功能的调控。H2AX 的 S139 磷酸化（形成 γH2AX）被认定为 DNA 双链断裂后最早发生的的事件之一，并作为 DNA 损伤修复蛋白的聚集点。7,8

组蛋白磷酸化也具有更广泛的功能：例如 H2B 磷酸化可促进细胞凋亡相关的染色质凝缩、DNA 片段化和细胞死亡。9

相关产品
抗 gamma H2A.X (phospho S139) | ChIP 级
抗组蛋白 H3 (phospho S10) 抗体 - Mitosis Marker
组蛋白 H3 (phospho S10) 检测试剂盒
组蛋白泛素化
组蛋白 H2A 和 H2B 是细胞核中泛素化程度最高的两种蛋白。10其中最常见的形式是 H2A 的 K119 单泛素化以及 H2B 的 K123（酵母）/K120（脊椎动物）单泛素化。但是，目前也发现了多泛素化的组蛋白，例如 H2A 和 H2AX 的 K63 多泛素化。

​​​​H2A 的单泛素化由多梳类蛋白催化，且大部分与基因沉默有关。 酵母中Bre1是负责H2B单泛素化的主要的酶；哺乳动物中是其同源物RNF20/RNF40。与 H2A 不同，单泛素化的 H2B 与转录激活有关。与其他组蛋白修饰类似，H2A 和 H2B 的单泛素化是可逆的，并由组蛋白泛素连接酶和去泛素化的酶严密调控。

组蛋白泛素化在 DNA 损伤响应中具有核心意义：RNF8/RNF168 催化组蛋白 H2A/H2AX 的 K63 多泛素化，并为 RAP80 和其他 DNA 修复蛋白提供识别位点。组蛋白 H2A、H2B 和 H2AX 的单泛素化也发生在 DNA 双链的断裂位点。

修饰标记​

过去许多年中，表观遗传学修饰被认为是不可逆的；修饰标记在经历多次细胞分裂后依然能保持稳定。但是，研究发现这一过程是高度动态的，并且由一组特定的酶调控。这些表观遗传学调控因子可分为修饰蛋白、识别蛋白和去修饰蛋白。

· 表观遗传学修饰蛋白
诸如组蛋白乙酰转移酶 (HAT)、组蛋白甲基转移酶 (HMT/KMT)、蛋白精氨酸甲基转移酶 (PRMT) 以及激酶等酶负责向组蛋白添加表观遗传学标记。

· 表观遗传学识别蛋白
这类蛋白识别并结合由修饰蛋白建立的表观遗传学标记，从而确定其功能。其中包括含有布罗莫结构域、染色质域和 Tudor 域的蛋白。

· 表观遗传学去修饰蛋白
去修饰蛋白，例如组蛋白去乙酰化酶 (HDAC)、赖氨酸去甲基化酶 (KDM) 和磷酸酶，催化表观遗传学标记的去除。

参考文献

1. Greer, E. L. & Shi, Y. Histone methylation:a dynamic mark in health, disease and inheritance. Nat. Rev. Genet. 13, 343–57 (2012).

2. Roth, S. Y., Denu, J. M. & Allis, C. D. Histone acetyltransferases. Annu. Rev. Biochem. 70, 81–120 (2001).

3. Nowak, S. J. & Corces, V. G. Phosphorylation of histone H3:A balancing act between chromosome condensation and transcriptional activation.Trends Genet. 20, 214–220 (2004).

4. Rossetto, D., Avvakumov, N. & Côté, J. Histone phosphorylation:A chromatin modification involved in diverse nuclear events.Epigenetics7, 1098–1108 (2012).

5. Banerjee, T. & Chakravarti, D. A Peek into the Complex Realm of Histone Phosphorylation. Mol. Cell. Biol. 31, 4858–4873 (2011).

6. Kschonsak, M. & Haering, C. H. Shaping mitotic chromosomes:From classical concepts to molecular mechanisms.BioEssays 755–766 (2015).

7. Lowndes, N. F. & Toh, G. W.-L. DNA repair:the importance of phosphorylating histone H2AX.Curr.Biol. 15, R99–R102 (2005).

8. Pinto, D. M. S. & Flaus, A. Structure and function of histone H2AX.Subcell.Biochem. 50, 55–78 (2010).

9. Füllgrabe, J., Hajji, N. & Joseph, B. Cracking the death code:apoptosis-related histone modifications.Cell Death Differ. 17, 1238–1243 (2010).

10. Cao, J. & Yan, Q. Histone ubiquitination and deubiquitination in transcription, DNA damage response, and cancer. Front. Oncol. 2, 26 (2012).

自从更新到Win10系统后，我的爱机X220就失去蓝牙功能了。网上搜索了许多解决办法，都没能凑效，今天终于解决了！我的蓝牙回归了，可以考虑买个蓝牙耳塞听听了。

方法献上，为更多苦恼的朋友解决难题。参照下面这个帖子。

https://answers.microsoft.com/en-us/windows/forum/windows_10-networking/how-to-turn-on-the-bluetooth-in-windows-10-on/64dc140c-2209-4d84-872d-bbf5a11f0910

I have fixed the missing bluetooth device on windows 10 with my Lenovo X220i Laptop by upgrading the BIOS from the Lenovo Support Page to the latest.

http://support.lenovo.com/cz/cs/products/Laptops-and-netbooks/ThinkPad-X-Series-laptops/ThinkPad-X220i/downloads/DS018805

The bluetooth device fired up instantly after doing so.

Try upgrading your BIOS of your machine to the latest.

Mine worked fine even though the BIOS upgrade utility does not list Windows 10 as supported OS. It worked just fine.

上面这位先生是在X220i机子上通过升级BIOS实现的。按照他的方法，我把X220的BIOS升级到最新的，Bluetooth立马出来了。我用的BIOS升级程序也不是Win10的，官方现在为止我没找到Win10的，我就用Win8 64bit的BIOS升级程序同样起作用了。

准备淘蓝牙耳机了！

希望能帮到需要的人。

June/July 2003

The design of siRNAs and short hairpin siRNAs (shRNAs) remains an empirical process since the molecular mechanisms underlying RNAi are not yet sufficiently understood to allow for the rational design of siRNAs. However, based on the research from various laboratories including our own, InvivoGen has been able to develop siRNA Wizard, an online tool accessible from our homepage, that will help you find the best siRNA sequences on your target gene. The siRNA Wizard tool will also design the pair of oligonucleotides needed to generate shRNAs using InvivoGen's psiRNA plasmids. Below is the list of general rules, used by the siRNA Wizard, that have been revised to better suit the design of shRNAs.

The current guidelines recommend avoiding the first 50-100 nt located downstream of the Start codon and the 100 nt located upstream of the Stop codon, as well as 5' and 3'UTRs. These regions contain binding sequences for regulatory proteins that may affect the accessibility of the RNA target sequence to the RISC complex. However, we and others have successfully silenced the expression of several genes by targeting the 5' or 3'UTRs [1, 2, 3, 4]. Therefore, 5' and 3'UTRs should also be considered when selecting a region on your target gene.

The first nucleotide of the siRNA sequence can either be an A or a G. Although we recommend choosing an A (see Selection criteria for Standard search), a G can also be used since in several examples siRNAs starting with a G and expressed from the human H1 promoter have worked [4, 5].
The pyrimidines C and T should be avoided because expression of RNAs from RNA polymerase III promoters is only efficient when the first transcribed nucleotide is a purine. In cases where your siRNA sequence starts with a C or T, we recommend adding an A as the first nucleotide.
This addition will not affect the activity of your siRNA since it will generate a T at the end of the antisense siRNA strand that will be included in the termination signal maintaining its complementarity with the target sequence. This point is important since according to current knowledge recognition of the specific gene target is achieved by the antisense siRNA strand.
It is usually recommended to choose sequences with low GC content (between 30-55%). There are also many examples of active siRNAs with high GC content [6, 7, 8].

siRNA-mediated RNAi is based on using dsRNA < 30 nt to avoid nonspecific silencing. According to Hannon et al. siRNA of 25-29 nt are generally more effective than shorter ones. However, we and others found that hairpin siRNAs with duplex length of 19-21 nt are as effective as longer hairpin siRNAs [6, 9, 10].

Several teams including ours have tested a variety of sequences for the loop between the two complementary regions of a shRNA, ranging from 3 to 9 nt in length. Similar effectiveness have been obtained for loops of 5, 7 or 9 nt. We use a 7 nt loop sequence (TCAAGAG) for the psiRNA vectors.

Despite the fact that this set of rules is still not well defined, sequences generated by the siRNA Wizard will likely work better than randomly selected sequences. However, because some candidate siRNAs are more active than others, it is recommended to vary the selection criteria and to compare a panel of three siRNAs to find the most efficient.

1- Yokota T. et al., 2003. EMBO reports AOP.
2- Yu JY. et al., 2002. PNAS 99(9):6047-6052
3- Rubinson DA. et al., 2003. Nature Genetics 33:401-406
4- Mcmanus MT. et al., 2002. RNA 8:842-850
5- Tiscornia G. et al., 2003. PNAS 100(4):1844-1848
6- Kim MH. et al., 2002. BBRC 296:1372-1377
7- Hasuwa H. et al., 2002. FEBS Letters 532:227-230
8- Bertrand JR. et al., 2002. BBRC 296:1000-1004
9- Yu JY. et al., 2003. Molecular Therapy 7(2):228-236
10- Song E. et al., 2003. Nature Medicine 9(3): 347-351

Reference from http://www.invivogen.com/review-sirna-shrna-design

Cells growing in log phase are the most suitable one for studying gene silencing because once the cells come to be in confluence, cell cycle will be blocked, senescence will start and a group of inhibins will be secreted. All these will affect the physiological status of the cells and in turn affect gene silencing.

I am assuming that the protein of interest is not density- or cell-cycle-dependent, otherwise the experimental needs are dictated by the exact question you are addressing. A "generic" protocol very much depends on your cells' doubling time and the time-frame of the physiological effect you expect, starting with the stability of your protein of interest. A fast-growing cell line (say, doubling time of 16-30 hrs) is best transfected at ~60-70% and analysed 24 hrs post transfection. BUT if your protein is stable (e.g. structural component), 24 hours may not be sufficient for its degradation, so starting with a sparser culture (~40%) and waiting 48 hours might be better. GFP protein, for example has a half life of several days! In a slow-cycling cell line, 80-90% is a good density.
The most robust approach is to try the knock down in parallel in dishes with different density first, choose a set of conditions, and stick to them.

Its good to avoid complete confluency at the time you harvest your cells, best would be 70-80% at harvesting time. So,dependent on the time you have to give the siRNA to work (could be 24,48,72 or more h) you will have to seed the amount of cells that ends up with 70-80% of cells at the harvesting time. Depends on the division time of your cell line. Design a test experiment, (differnt KD times, cell# etc.. ) and give it a try.

Confluent monolayer is not a good thing when working with eukaryotic cells. If my cells reach confluence I am forced to eliminate them. So in theory, the cells on plastic should never reach confluence, especially if you are going to freeze them and use as stocks.

A more convenient and efficient way of transfecting cells is reverse-transfection. You detach the cells from plastic and transfect them in solution and then add medium and aliquot to the tissue culture plates. This is a more robust and high-throughput method compared to just adding your transfection mix to cells growing on plastic and embedded in extracellular matrix (I am not talking here about suspension cell cultures as these are a special case).

For siRNA knock-down, 24 hours is almost never enough to see 1-2 log difference. You need 48 hours, at least, to get a big difference.

In principle, cell confluency per se brings up stress to the cell population and is able to activate signaling pathways (like NF-kB) and, if the process is persistent, can certainly establish some selective pressure for the cells. Depending on the target gene you are focusing on, your gene silencing strategy might be affected when cells are under this kind of stress. Besides, compensatory effects frequently happen when stably shRNA expressing cells are cultured for long time (better to freeze stocks and culture cells for only 2-3 passages)

You no need to seed cells at 80 - 90% confluency, there is difference between knock-down and over-expression.

For silencing, transfection at 30 - 40 % is more efficient than higher confluency, We usually do knockdown for 72 hrs (for smaller proteins) and 90 hrs (for larger proteins).

Again, it depends on how efficient the siRNA is targeting the mRNA. In my experience, once the siRNA is taken up by the cells (usually within 8hrs post-trans) it is very stable inside.

PS: I did knock-down for a protein, 72 hours post-trans, I transfected its deletion mutant (only C-Terminus region) to see the effect (after 36 hours). But, the siRNA was still active and it silenced my deletion mutant also.

Good idea from https://www.researchgate.net/post/Which_is_better_for_studying_siRNA_miRNA_silencing_a_growing_cell_layer_or_a_confluent_monolayer

If PCR is performed using a proofreading DNA polymerase, such as Pfu DNA polymerase, the product will have blunt ends. Taq DNA polymerase catalyzes the non-template directed addition of an adenine residue to the 3´-end of both strands of DNA molecules to make it suitable for TA cloning.

Details: http://openwetware.org/wiki/Addition_of_3'_A_overhangs_to_PCR_products