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8062
Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (PE Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (PE Conjugate) #8062

Citations (7)
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Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with hIFN-α1 #8927 (100 ng/mL, 5 min; green) using Phospho-Stat1 (Tyr701) (58D6) (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).
To Purchase # 8062S
Cat. # Size Price Inventory
8062S
100 µl  (50 tests)

Supporting Data

REACTIVITY H M
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 cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167.

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 antibodies. 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 Stat1 在 Tyr701 位点被磷酸化时,Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (PE Conjugate) 才能识别出内源水平的 Stat1 。该抗体可检测在 Tyr701 位点被磷酸化的 Stat1α 和 Stat1β。它不会与其他 Stat 蛋白的相应磷酸酪氨酸发生交叉反应。

物种反应性:

人, 小鼠

来源/纯化

使用与人 Stat1 蛋白中 Tyr701 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

在有大量配体的情况下,Stat1 转录因子被激活 (1),并且对 IFN-α 和 IFN-γ 反应非常重要 (2,3)。Stat1的 Tyr701 位点磷酸化会诱导 Stat1 二聚化、核转位和 DNA 结合 (4)。Stat1 蛋白有一对同工型:Stat1α (91 kDa) 和剪切变体 Stat1β (84 kDa)。在多数细胞中,两种同工型被 IFN-α 激活,但仅 Stat1α 被 IFN-γ 激活。许多肿瘤细胞会出现不正常的 Stat1 激活 (5)。除了酪氨酸磷酸化,Stat1的 Ser727位点还能在 IFN-α 和其他细胞应激的刺激下通过 p38 丝裂原活化蛋白激酶 (MAPK) 依赖的通路被磷酸化 (6)。Stat1 介导的基因激活的最大诱导可能需要丝氨酸磷酸化。

通路

探索与本品相关的通路。

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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