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92109
Phospho-ATF-2 (Thr71)/ATF-7 (Thr53) (A8J7P) Rabbit mAb (PE Conjugate)
抗体偶联物
单克隆抗体

Phospho-ATF-2 (Thr71)/ATF-7 (Thr53) (A8J7P) Rabbit mAb (PE Conjugate) #92109

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Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with Anisomycin #2222 (25 ug/ml, 30 min; green), using Phospho-ATF-2 (Thr71)/ATF-7 (Thr53) (A8J7P) Rabbit mAb (PE Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).
To Purchase # 92109S
Cat. # Size Price Inventory
92109S
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 Phospho-ATF-2 (Thr71)/ATF-7 (Thr53) (A8J7P) Rabbit mAb #15411.

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

Phospho-ATF-2 (Thr71)/ATF-7 (Thr53) (A8J7P) Rabbit mAb (PE Conjugate) 可检测仅在苏氨酸 71 和苏氨酸 53 上分别被磷酸化的 ATF-2 和 ATF-7 的内源水平。该抗体不与磷酸化的 c-Jun、CREB 或其他转录因子发生交叉反应。它可同样好地识别 Thr69/Thr71 双磷酸化的 ATF-2、Thr51/Thr53 双磷酸化的 ATF-7、Thr71 单磷酸化 ATF-2 和 Thr53 单磷酸化 ATF-7。

物种反应性:

人, 小鼠, 大鼠, 猴

来源/纯化

使用与人 ATF-2 蛋白中 Thr71 周围残基相对应的合成磷酸肽,对动物进行免疫接种来产生单克隆抗体。

背景

转录因子 ATF-2(也被称为 CRE-BP1)结合 AP-1 和 CRE DNA 效应元件,同时也是亮氨酸拉链蛋白家族 ATF/CREB 的一员 (1)。ATF-2 与各种病毒癌蛋白以及细胞抑癌基因相互作用,并且是 SAPK/JNK 和 p38 MAP 激酶信号转导通路的靶标 (2-4)。各种细胞应激(包括基因毒剂、炎性细胞因子和紫外辐射)都能刺激 ATF-2 的转录活性。细胞应激通过在 Thr69 和 Thr71 的磷酸化激活 ATF-2 (2-4)。在体内和经 ATF-2 转染的细胞内,SAPK 和 p38 MAPK 能在这些位点磷酸化 ATF-2。这些位点的突变导致应激诱导的 ATF-2 转录缺失 (2-4)。此外,这些位点的突变会降低 E1A 和 Rb 通过ATF-2 刺激基因表达的能力 (2)。

通路

探索与本品相关的通路。

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。

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仅供研究使用。不得用于诊断流程。
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