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14609
c-Fos (9F6) Rabbit mAb (PE Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

c-Fos (9F6) Rabbit mAb (PE Conjugate) #14609

Citations (2)
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Flow cytometric analysis of HeLa cells, untreated (blue) or treated with TPA #4174 (400 nM, 4 hr; green), using c-Fos (9F6) Rabbit mAb (PE Conjugate).
To Purchase # 14609S
Cat. # Size Price Inventory
14609S
100 µl  (50 tests)

Supporting Data

REACTIVITY H M R
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 c-Fos (9F6) Rabbit mAb #2250.

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

c-Fos (9F6) Rabbit mAb (PE Conjugate) 可识别内源水平的 c-Fos 总蛋白。该抗体不会与 FosB、FRA1 或 FRA2 等其他 Fos 蛋白发生交叉反应。

物种反应性:

人, 小鼠, 大鼠

基于 100% 序列同源性预测发生反应的物种:

仓鼠 , 牛 , 猪

来源/纯化

使用与人 c-Fos 蛋白中氨基末端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

胞核癌基因 Fos 家族包括 c-Fos、FosB、Fos-相关抗原 1 (FRA1) 和 Fos-相关抗原 2 (FRA2) (1)。虽然大部分 Fos 蛋白作为单一亚型存在,但是 FosB 蛋白作为两种亚型存在:全长 FosB 和缩短形式 FosB2 (δ FosB),后者缺乏羧基端的 101 个氨基酸 (1-3)。Fos 蛋白的表达可由各种胞外刺激快速且瞬间诱导,这些刺激包括生长因子、细胞因子、神经递质、多肽激素和应激反应。Fos 蛋白与 Jun 蛋白(c-Jun、JunB 和 JunD)二聚化以形成激活蛋白-1 (AP-1),一种与 TRE/AP-1 元件结合并激活转录的转录因子。Fos 蛋白和 Jun 蛋白包含可介导二聚化的亮氨酸拉链基序和一个与 DNA 结合的毗邻碱性结构域。各种 Fos/Jun 异二聚体在反式激活 AP-1 依赖性基因方面的能力不同。Erk 激酶引起的 Fos 蛋白磷酸化对胞外刺激应答后,除了表达增加外,还可能会进一步增强转录活性 (4-6)。Erk5 引起的 c-Fos 在 Ser32 和 Thr232 的磷酸化增强了蛋白质稳定性及核定位 (5)。Erk1/2 引起的 FRA1 在 Ser252 和 Ser265 的磷酸化增强了蛋白质稳定性并导致 FRA1 在癌细胞中过量表达 (6)。生长因子刺激后,FosB 和 c-Fos 的表达在静息成纤维细胞中立即开始,但是持续时间很短,蛋白水平在几小时后便消失了 (7)。FRA1 和 FRA2 表达持续的时间更长,并且可以在非同步生长的细胞中检测到它的相对水平 (8)。去调控的 c-Fos、FosB 和 FRA2 表达可能导致肿瘤性细胞转化;但 δ FosB 不能转化细胞 (2,3)。

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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