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5123
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Alexa Fluor® 647 Conjugate)
抗体偶联物
单克隆抗体

Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Alexa Fluor® 647 Conjugate) #5123

Citations (6)
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Flow cytometric analysis of jurkat cells, untreated (green) or treated with LY294002 #9901 (50uM, 2hr), Wortmannin #9951 (1uM, 2hr), and U0126 #9903 (10uM, 2hr) (blue), using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Alexa Fluor® 647 Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (dashed lines).
To Purchase # 5123S
Cat. # Size Price Inventory
5123S
100 µl  (50 tests)

Supporting Data

REACTIVITY H M R Mk Dm
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 (CST) antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometric analysis of human cells. The unconjugated antibody, Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855, reacts with Phospho-4E-BP1 (Thr37/46) from human, mouse, rat and monkey. CST expects that phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Alexa Fluor® 647 Conjugate) will also recognize Phospho-4E-BP1 in these species.

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 4E-BP1 在 Thr37 和/或 Thr46 位点被磷酸化时,Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Alexa Fluor® 647 Conjugate) 方可检测内源水平的 4E-BP1 。该抗体可能会与在相同位点被磷酸化的 4E-BP2 和 4E-BP3 发生交叉反应。

物种反应性:

人, 小鼠, 大鼠, 猴, 黑腹果蝇

来源/纯化

使用与小鼠 4E-BP1 中 Thr37 和 Thr46 周围的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。该抗体在最合适的条件下偶联 Alexa Fluor® 647,其中 F/P 比值为 2-5。
红光(如 633 nm 氦氖激光)可最大程度刺激 Alexa Fluor® 647 染料。Alexa Fluor® 647 染料的抗体接合物会产生明亮的远红外荧光发射,峰值为 665 nm。

背景

翻译抑制蛋白 4E-BP1(也称为 PHAS-1)可通过结合翻译起始因子 eIF4E 抑制帽子结构依赖性翻译。4E-BP1 的高度磷酸化会扰乱这种相互作用,进而导致帽子结构依赖性翻译被激活 (1)。PI3 激酶/Akt 通路和 FRAP/mTOR 激酶均可调节 4E-BP1 活性 (2,3)。多个 4E-BP1 残基在体内被磷酸化 (4)。一般认为FRAP/mTOR 在 Thr37 和 Thr46 的磷酸化虽然不会阻碍 4E-BP1 与 eIF4E的结合,但会引导4E-BP1 后续在 Ser65 和 Thr70 的磷酸化 (5)。

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。

产品用“仅供研究使用”或类似标示声明标示,并且尚未经 FDA 或其他国外或国内监管实体出于任何目的批准、准许或许可。客户不得出于任何诊断或治疗目的或以任何与产品标示声明相冲突的方式使用任何产品。CST 销售或许可的产品提供给作为最终用户的客户,且仅用于研究和开发用途。出于诊断、预防或治疗目的任何产品使用或出于转售(单独或作为成分)或其他商业目的的任何产品购买都要求来自 CST 的单独许可。客户 (a) 不得向任何第三方出售、许可、出借、捐赠或另行转让或提供任何本公司产品,无论单独或联合其他材料方式,或使用本公司产品制造任何商业产品,(b) 不得复制、修改、逆向工程、反编译、反汇编或另行尝试发现本公司产品的底层结构或技术,或出于开发与 CST 产品或服务竞争的任何产品或服务的目的使用本公司产品,(c) 不得从本公司产品改变或移除任何商标、商品名称、徽标、专利或版权声明或标记,(d) 仅应根据 CST 产品销售条款和任何适用文档使用本公司产品,以及 (e) 应就客户联系本公司产品所用的任何第三方产品或服务而言遵守任何许可、服务条款或类似协议。

仅供研究使用。不得用于诊断流程。
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