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65221
Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb (PE Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb (PE Conjugate) #65221

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Flow cytometric analysis of HeLa cells, untreated (blue) or treated with Vorinostat (SAHA) #12520 (2 μM, 16 hr; green), using Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb (PE Conjugate) (solid lines) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).
To Purchase # 65221S
Cat. # Size Price Inventory
65221S
100 µl  (50 tests)

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb #12799.

Product Usage Information

Application Dilution
Flow Cytometry (Fixed/Permeabilized) 1:50

Storage

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised June 2020

实验步骤编号:407

特异性/灵敏度

Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAb (PE Conjugate) 可检测仅在 Lys5 被乙酰化的组蛋白 H2B 的内源水平。该抗体不与其他乙酰化组蛋白发生交叉反应。

物种反应性:

人, 小鼠, 大鼠, 猴

基于 100% 序列同源性预测发生反应的物种:

仓鼠 , 鸡 , 斑马鱼 , 牛 , 马

来源/纯化

使用与人组蛋白 H2B 蛋白的乙酰化 Lys5 周围的残基相对应的合成肽对动物进行免疫接种来产生单克隆抗体。

背景

核小体由四个核心组蛋白(H2A、H2B、H3 和 H4)组成,是染色质的主要组成部分。组蛋白最初被视为作为 DNA 包装的静态支架蛋白起作用,但现在已被证明是一种经过多种类型的翻译后修饰的动态蛋白,包括乙酰化、磷酸化、甲基化和泛素化 (1,2)。在转录激活期间,p300/CBP 组蛋白乙酰转移酶在基因启动子上乙酰化组蛋白 H2B 氨基末端尾区的多个赖氨酸残基(Lys5、12、15 和 20)(1-3)。组蛋白尾区的过度乙酰化会中和这些结构域的正电荷,弱化组蛋白-DNA 和核小体之间的相互作用,因此会导致染色质结构不稳定,增加进入不同 DNA 结合蛋白的 DNA (4,5)。此外,特定赖氨酸残基的乙酰化会形成锚定位点,有助于募集包含溴结构域的许多转录和染色质调节蛋白,进而结合乙酰化赖氨酸残基 (6)。在转录激活期间,RAD6 E2 蛋白和 BRE1A/BRE1B E3 连接酶(也称为 RNF20/RNF40)在 Lys120 位点单泛素化组蛋白 H2B (7)。单泛素化组蛋白 H2B Lys120 与激活基因的转录区域有关,并通过促进 FACT 依赖性染色质重构刺激转录延长 (7-9)。此外,组蛋白 H3 Lys4 和 Lys79 的后续甲基化也很重要,因为这是调节转录起始和延长的其他两种组蛋白修饰 (10)。在代谢应激的刺激下,AMPK 被募集到应答基因,并在启动子和基因转录区域的 Lys36 位点磷酸化组蛋白 H2B,进而调节转录延长 (11)。在多种细胞凋亡刺激物的刺激下,Mst1 激酶磷酸化组蛋白 H2B 的 Ser14 位点 (12)。在诱导细胞凋亡后,caspase-3 裂解和激活 Mst1,导致组蛋白 H2B 在染色质凝聚期间被全部磷酸化。有趣的是,组蛋白 H2B 在小鼠胚胎成纤维细胞的辐射诱导性 DNA 损伤基因座中被快速磷酸化 (13)。在此情况下,根据 H2AX Ser139 之前的磷酸化,Ser14 位点的磷酸化十分迅速,并且在凋亡不存在的情况下发生,表明 Ser14 磷酸化可能在 DNA 损伤修复和细胞凋亡中起明显作用。

有限使用

除非如以 CST 合法授权代表签署的书面形式另行明确同意,否则以下条款适用于 CST、其附属公司或其分销商提供的产品。除非 CST 合法授权代表以书面形式单独接受,否则任何附加于或异于此处所载条款和条件的客户条款和条件均被拒绝且无效。

产品用“仅供研究使用”或类似标示声明标示,并且尚未经 FDA 或其他国外或国内监管实体出于任何目的批准、准许或许可。客户不得出于任何诊断或治疗目的或以任何与产品标示声明相冲突的方式使用任何产品。CST 销售或许可的产品提供给作为最终用户的客户,且仅用于研究和开发用途。出于诊断、预防或治疗目的任何产品使用或出于转售(单独或作为成分)或其他商业目的的任何产品购买都要求来自 CST 的单独许可。客户 (a) 不得向任何第三方出售、许可、出借、捐赠或另行转让或提供任何本公司产品,无论单独或联合其他材料方式,或使用本公司产品制造任何商业产品,(b) 不得复制、修改、逆向工程、反编译、反汇编或另行尝试发现本公司产品的底层结构或技术,或出于开发与 CST 产品或服务竞争的任何产品或服务的目的使用本公司产品,(c) 不得从本公司产品改变或移除任何商标、商品名称、徽标、专利或版权声明或标记,(d) 仅应根据 CST 产品销售条款和任何适用文档使用本公司产品,以及 (e) 应就客户联系本公司产品所用的任何第三方产品或服务而言遵守任何许可、服务条款或类似协议。

仅供研究使用。不得用于诊断流程。
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