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94805
Bim (C34C5) Rabbit mAb (Alexa Fluor® 488 Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

Bim (C34C5) Rabbit mAb (Alexa Fluor® 488 Conjugate) #94805

Citations (3)
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  1. F
Flow cytometric analysis of HeLa cells using Bim (C34C5) Rabbit mAb (Alexa Fluor® 488 Conjugate) (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (red).
To Purchase # 94805S
Cat. # Size Price Inventory
94805S
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 Alexa Fluor® 488 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 Bim (C34C5) Rabbit mAb #2933.

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

Bim (C34C5) Rabbit mAb (Alexa Fluor® 488 Conjugate) 可检测内源水平的 Bim 总蛋白(EL、L 和 S 同工型)。

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:

猴, 牛 , 犬 , 猪

来源/纯化

使用与 Bim Pro25 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

Bim/Bod 是一种促凋亡蛋白,属于 Bcl-2 家族中仅包含 BH3 的亚家族,该亚家族成员包括 Bad、Bid、Bik、Hrk 和 Noxa,都包含一个 BH3 结构域,但缺乏其他保守的 BH1 或 BH2 结构域 (1,2)。Bim 结合并拮抗 Bcl-2 家族的抗凋亡成员而诱导凋亡。研究观察到 Bim 与 Bcl-2、Bcl-xL、Mcl-1、Bcl-w、Bfl-1 和 BHRF-1 相互作用 (1,2)。Bim 可调节与胸腺细胞阴性选择和随后的生长因子退出有关的细胞凋亡,在生长因子退出过程中 Bim 表达升高 (3-6)。选择性剪切会产生三种主要的 Bim 同工型:BimEL、BimL 和 BimS (1)。BimS 是最短的同工型,细胞毒性最强,通常仅在细胞凋亡过程中瞬时表达。BimEL 和 BimL 同工型能通过与动力蛋白轻链相互作用被隔绝到动力蛋白运动复合体,并在细胞凋亡期间从这个复合体上释放 (7)。其余较长同工型的凋亡活性可能受到磷酸化调节 (8,9)。环境应激会引起 JNK 磷酸化 Bim,使 Bim 与动力蛋白复合体解离并增加凋亡活性。

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。
Cell Signaling Technology 是 Cell Signaling Technology, Inc. 的商标。
Alexa Fluor 是 Life Technologies 公司的注册商标。
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