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98789
Acetyl-Histone H4 (Lys16) (E2B8W) Rabbit mAb (Alexa Fluor® 647 Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

Acetyl-Histone H4 (Lys16) (E2B8W) Rabbit mAb (Alexa Fluor® 647 Conjugate) #98789

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Flow cytometric analysis of HeLa cells, untreated (blue) or treated with Trichostatin A (TSA) #9950 (1 μM, 18 hr; green), using Acetyl-Histone H4 (Lys16) (E2B8W) Rabbit mAb (Alexa Fluor® 647 Conjugate).
To Purchase # 98789S
Cat. # Size Price Inventory
98789S
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 Alexa Fluor® 647 fluorescent dye 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 H4 (Lys16) (E2B8W) Rabbit mAb #13534.

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

仅当组蛋白 H4 在 Lys16 位点被乙酰化时,Acetyl-Histone H4 (Lys16) (E2B8W) Rabbit mAb (Alexa Fluor® 647 Conjugate) 才能识别出内源水平的组蛋白 H4。这种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

来源/纯化

使用与人类 组蛋白 H4 的 Lys16 周围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)。组蛋白乙酰化主要在组蛋白 H2A (Lys5)、H2B(Lys5、12、15 和 20)、H3(Lys9、14、18、23、27、36 和 56)以及 H4(Lys5、8、12 和 16)的氨基末端尾结构域上发生,且对调节组蛋白沉积、转录激活、DNA 复制、重组和 DNA 修复而言至关重要 (1-3)。组蛋白尾的过度乙酰化可中和这些结构域的正电荷,并且被认为可削弱组蛋白-DNA 之间的相互作用和核小体-核小体间相互作用,因而使染色质结构去稳定化并且增加 DNA 对多种 DNA 结合蛋白的可及性 (4,5)。此外,特定赖氨酸残基的乙酰化产生称作溴结构域的蛋白质模块的对接位点,这种蛋白质模块与乙酰化赖氨酸残基结合 (6)。许多转录调节蛋白和染色质调节蛋白含有溴结构域并且可以被募集至基因启动子,部分可通过结合乙酰化的组蛋白尾部被募集。组蛋白乙酰化由组蛋白乙酰转移酶 (HAT) (如 CBP/p300、GCN5L2、PCAF 和 Tip60)介导,这些酶由 DNA 结合蛋白因子募集至基因,以促进转录激活 (3)。去乙酰化,由组蛋白脱乙酰基酶(HDAC 和沉默调节蛋白)介导,可逆转乙酰化的作用并且通常会促进转录抑制 (7,8)。

通路

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有限使用

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仅供研究使用。不得用于诊断流程。
Cell Signaling Technology 是 Cell Signaling Technology, Inc. 的商标。
Alexa Fluor 是 Life Technologies 公司的注册商标。
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