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16318
Phospho-SLP-76 (Ser376) (D7S1K) Rabbit mAb (PE Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

Phospho-SLP-76 (Ser376) (D7S1K) Rabbit mAb (PE Conjugate) #16318

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Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left column) or treated with cross-linked anti-CD3 plus anti-CD28 (10 μg/ml each, 15 min; right column), using Phospho-SLP-76 (Ser376) (D7S1K) XP® Rabbit mAb (PE Conjugate) (top row) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (bottom row), and co-stained with a CD3 antibody. 
To Purchase # 16318S
Cat. # Size Price Inventory
16318S
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 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-SLP-76 (Ser376) (D7S1K) Rabbit mAb #92711.

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 SLP-76 蛋白在 Ser376 位点被磷酸化时,Phospho-SLP-76 (Ser376) (D7S1K) XP® Rabbit mAb (PE Conjugate) 才能识别出内源水平的 SLP-76 蛋白。
通过流式细胞术检测克隆 E3G9U 比检测克隆 D7S1K 更敏感。

物种反应性:

来源/纯化

使用与人 SLP-76 蛋白中 Ser376 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

包含 SH2 结构域的 76 kDa 白细胞蛋白 (SLP-76) 是一种造血细胞接头蛋白,它对多个生化信号转导通路都非常重要,并且为 T 细胞发育和激活所必需 (1)。连接 TCR 会使 ZAP-70 磷酸化 SLP-76 和 LAT。SLP-76 的氨基末端酪氨酸残基后跟一个脯氨酸富集结构域和一个羧基末端 SH2 结构域。Tyr113 和 Tyr128 磷酸化会募集 GEF Vav 和接头蛋白 Nck (2)。连接 TCR 还会导致 Tyr145 磷酸化,从而介导 SLP-76 和 Itk 之间的结合,这在某种程度上通过 SLP-76 的脯氨酸富集结构域和 Itk 的 SH3 结构域完成 (3)。此外,SLP-76 的脯氨酸富集结构域结合类 Grb2 接头蛋白 Gads 的 SH3 结构域 (3,4)。在静息细胞中,SLP-76 主要在胞质中表达。在连接 TCR 后,SLP-76 转位到质膜,并促进多蛋白信号转导复合体的组装,该复合体包括 Vav、Nck、Itk 和 PLCγ1(1)。SLP-76 表达受严格调控;该蛋白在胸腺细胞发育的早期阶段可被检测到,随着胸腺细胞成熟过程的进行表达升高,并在细胞发育成成熟 CD4+ CD8+ 双阳性胸腺细胞的过程中表达降低 (5)。

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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