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44721
PKCα (D7E6E) Rabbit mAb (PE Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

PKCα (D7E6E) Rabbit mAb (PE Conjugate) #44721

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Flow cytometric analysis of SK-MEL-2 (blue) and SH-SY5Y (green) cells using PKCα (D7E6E) Rabbit mAb (PE Conjugate).
To Purchase # 44721S
Cat. # Size Price Inventory
44721S
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. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated PKCα (D7E6E) Rabbit mAb #59754.

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

PKCα (D7E6E) Rabbit mAb (PE Conjugate) 可识别内源水平的 PKCα 总蛋白。

物种反应性:

人, 小鼠, 大鼠

来源/纯化

使用与人 PKCα 蛋白中 Val664 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

在调控分泌、基因表达、细胞增殖和肌肉收缩等多个细胞反应的级联中,蛋白激酶 C (PKC) 激活是该级联中最早期的一个活动 (1,2)。根据钙依赖性和激活因子,PKC 同工型属于三个亚家族。典型 PKC 因其 C2 结构域对钙具有依赖性,可通过其半胱氨酸富集 C1 结构域被磷脂酰丝氨酸 (PS)、甘油二酯 (DAG) 和佛波酯 (TPA, PMA) 激活。新型和非典型 PKC 均有钙依赖性,但只有新型 PKC 才能被 PS、DAG 和佛波酯激活 (3-5)。这三个 PKC 亚家族的成员包含一个假底物或自抑制结构域,可结合催化结构域中的底物结合位点,以防止在没有辅因子或激活因子的情况下被激活。PKC 活性的控制通过三个独立的磷酸化事件进行调节。活化环中的 Thr500、Thr641(自磷酸化)以及羧基末端疏水性位点 Ser660 均会发生体内磷酸化 (2)。非典型 PKC 同工型缺乏疏水区域磷酸化,这与谷氨酸的存在有关,与在更典型的 PKC 同工型中发现的丝氨酸或苏氨酸残基无关。PDK1 或相关酶负责 PKC 的激活。PKC 超家族最近新增加的成员是 PKCμ (PKD),它通过其 C1 结构域被 DAG 和 TPA 调控。PKD 的特点是有一个 PH 结构域,它有独特的底物识别和高尔基体定位特征 (6)。PKC 相关激酶 (PRK) 缺少 C1 结构域,并且不对 DAG 或佛波酯产生反应。磷脂酰肌醇脂质激活 PRK,小 Rho 家族 GTP 酶结合同源区域 1 (HR1),可调节 PRK 激酶活性 (7)。

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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