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64137
Phospho-Btk (Tyr551) (E5Y6N) Rabbit mAb (Alexa Fluor® 647 Conjugate)
抗体偶联物
单克隆抗体

Phospho-Btk (Tyr551) (E5Y6N) Rabbit mAb (Alexa Fluor® 647 Conjugate) #64137

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Flow cytometric analysis of Ramos cells, untreated (blue) or treated with IgM (12 μg/ml, 10 min; green), using Phospho-Btk (Tyr551) (E5Y6N) 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 # 64137S
Cat. # Size Price Inventory
64137S
100 µl  (50 tests)

Supporting Data

REACTIVITY H
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® 647 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 Phospho-Btk (Tyr551) (E5Y6N) Rabbit mAb #18805.

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-Btk (Tyr551) (E5Y6N) Rabbit mAb(Alexa Fluor® 647 Conjugate)仅在 Tyr551 位点磷酸化时才可识别 Btk 蛋白的内源水平。

物种反应性:

来源/纯化

使用与人 Btk 蛋白中 Tyr551 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

Bruton 酪氨酸激酶 (Btk) 是细胞质酪氨酸激酶 Btk/Tec 家族的成员。与其他 Btk 家族成员一样,它含有一个 pleckstrin 同源性 (PH) 结构域以及 Src 同源性 SH3和 SH2 结构域。Btk 在 B 细胞发育中发挥重要作用 (1,2)。各种配体激活 B 细胞的同时,Btk 膜转位受到其 PH 结构域与磷脂酰肌醇-3,4,5-三磷酸结合的介导 (3-5)。Btk 定位于细胞膜后具有活性并瞬间磷酸化两个酪氨酸残基 Tyr551 和 Tyr223激活环中的 Tyr551 位点被 Src 家族酪氨酸激酶转磷酸化导致在 SH3 结构域内的 Tyr223 位点的自磷酸化,这对 B 细胞的完全激活非常重要 (6,7)。PKCβ 通过磷酸化 Btk 的 Ser180 位点而负向调控 Btk 的激活,并导致了细胞膜招募能力降低、磷酸根转移和后续的激活8。PKC 抑制信号可能是 B 细胞受体信号转导阈值维持最佳 Btk 活性的重要决定因素 (8)。

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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