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11849
PAX5 (D19F8) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)
抗体偶联物
单克隆抗体

PAX5 (D19F8) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #11849

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Flow cytometric analysis of Jurkat cells (blue) and Ramos cells (green) using PAX5 (D19F8) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines).
To Purchase # 11849S
Cat. # Size Price Inventory
11849S
100 µl  (50 tests)

Supporting Data

REACTIVITY H M
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 cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated PAX5 (D19F8) XP® Rabbit mAb #8970.

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

PAX5 (D19F8) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) 可识别内源水平的 PAX5 总蛋白。

物种反应性:

人, 小鼠

基于 100% 序列同源性预测发生反应的物种:

非洲爪蟾蜍

来源/纯化

使用与人 PAX5 蛋白中 Gln350 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

成对盒 (PAX) 蛋白是一个转录因子家族,在动物发育中起着不同的重要作用 (1)。现已在人和其他哺乳动物细胞中发现了 9 种 PAX 蛋白 (PAX1-9)。它们存在一个氨基末端“成对”结构域,该结构域包括两个螺旋-转角-螺旋基序,并且有 DNA 结合活性 (2)。根据是否存在一个羧基末端同源结构域和一个中央八肽结构域,PAX 蛋白分为四个结构相异的亚家族 (I-IV)。亚家族 I(PAX1 和 9)包含八肽结构域,但缺乏同源结构域;亚家族 II(PAX2、5 和 8)包含八肽结构域和一个截短同源结构域;亚家族 III(PAX3 和 7)包含八肽结构域和一个完整的同源结构域;亚家族 IV(PAX4 和 6)包含一个完整的同源结构域,但缺乏八肽结构域 (2)。PAX 蛋白可调节负责胚胎成型和器官形成的转录网络,因而在发育中发挥极其重要的作用 (3);部分 PAX 蛋白对出生后的发育也具有重要的作用 (4)。研究表明,会导致 PAX 基因异常表达的基因突变与许多癌症亚型有关 (1-3),其中亚家族 II 和 III 的成员被发现是肿瘤进展的潜在调节因子 (2)。

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。
XP 是 Cell Signaling Technology, Inc. 的注册商标。
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