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49236
Histone H2A.X (D17A3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

Histone H2A.X (D17A3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #49236

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Flow cytometric analysis of U-937 cells (blue) and MCF7 cells (green) using Histone H2A.X (D17A3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (dashed lines).
To Purchase # 49236S
Cat. # Size Price Inventory
49236S
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 Histone H2A.X (D17A3) XP® Rabbit mAb #7631.

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

Histone H2A.X (D17A3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate)可识别内源水平的总 histone H2A.X 蛋白。抗体不会与其他 H2A 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 histone H2A.X 蛋白的 Val124 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

组蛋白 H2A.X 是一种组蛋白变体,约占正常人成纤维细胞中总 H2A 组蛋白的 10% (1)。H2A.X 对检查点介导的细胞周期停滞、DNA 双链断裂后的 DNA 修复必不可少 (1)。电离辐射、紫外线灯或拟辐射剂所致 DNA 损伤,可导致 H2A.X 在 Ser139 位点被 PI3K 样激酶(包括 ATM、ATR 和 DNA-PK )快速磷酸化(2,3)。DNA 损伤后几分钟内,在 DNA 损伤位置的H2A.X Ser139 位点发生磷酸化 (4)。在 DNA 损伤反应中的这一极早期事件中,募集大量 DNA 损伤反应蛋白必不可少,其中包括有 MDC1、NBS1、RAD50、MRE11、53BP1 和 BRCA1 (1)。除了在 DNA 损伤修复中发挥作用外,H2A.X 对凋亡过程中 DNA 碎裂也不可或缺,并且在对凋亡信号作出响应时,会由不同的激酶磷酸化。在细胞死亡受体激活时,H2A.X  由 DNA-PK 在 Ser139 位点磷酸化,在长波紫外线作用下,由 c-Jun 氨基末端激酶 (JNK1) 在 Ser139 位点磷酸化,在血清饥饿时,由 p38 MAPK 在 Ser139 位点磷酸化 (5-8)。H2A.X 在未损伤细胞中由 WSTF 在 Tyr142 位点持续磷酸化(Williams-Beuren 综合征转录因子)(9,10)。在 DNA 损伤基础上,同时发生 Ser139 磷酸化,Tyr142 通过募集 EYA1 和 EYA3 磷酸酶在 DNA 损伤位点发生去磷酸化 (9)。虽然 Ser139 位点的磷酸化促进 DNA 修复蛋白和凋亡蛋白募集到 DNA 损伤位点,但 Tyr142 位点的磷酸化似乎可决定募集哪些蛋白。H2A.X 在 Tyr142 位点的磷酸化抑制 DNA 修复蛋白的募集,促进促凋亡因子如 JNK1 的结合 (9)。小鼠胚胎成纤维细胞只表达 H2A.X Y142F 突变体,此突变体倾向于 DNA 修复蛋白的募集胜过凋亡蛋白,表现出对电离辐射的凋亡反应减少 (9)。因此,H2A.X 在 Tyr142 的磷酸化和去磷酸化平衡,是 DNA 损伤后的细胞转归的转换机制。

有限使用

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仅供研究使用。不得用于诊断流程。
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