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63371
HSF1 (D3L8I) Rabbit mAb (PE Conjugate)
抗体偶联物
单克隆抗体
R
Recombinant

HSF1 (D3L8I) Rabbit mAb (PE Conjugate) #63371

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Flow cytometric analysis of HeLa cells using HSF1 (D3L8I) Rabbit mAb (PE Conjugate) (blue) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (red).
To Purchase # 63371S
Cat. # Size Price Inventory
63371S
100 µl  (50 tests)

Supporting Data

REACTIVITY H M R Mk B Dg Pg
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 HSF1 (D3L8I) Rabbit mAb #12972.

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

HSF1 (D3L8I) Rabbit mAb (PE Conjugate) 可识别内源水平的 HSF1 总蛋白。预计该抗体不会与 HSF2 发生交叉反应。

物种反应性:

人, 小鼠, 大鼠, 猴, 牛 , 犬 , 猪

来源/纯化

使用与人 HSF1 蛋白羧基末端附近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

在温度升高和其他环境应激的刺激下,所有生物体均可迅速诱导高度保守的热休克蛋白 (HSP) 的表达以作出反应,HSP 可作为分子伴侣重新折叠变性蛋白并促进受损蛋白的降解。热休克基因转录通过热休克因子 (HSF) 家族调节,HSF 是一种转录激活因子,可结合位于所有热休克基因上游的热休克反应元件 (HSE) (1)。HSE 在生物体中高度保守,包含五核苷酸基序 5'-nGAAn-3' 的多个相邻和反向重复序列。HSF 的保守性稍差,各 HSF 之间仅有 40% 的序列同源性。脊椎动物细胞包含 4 个 HSF 蛋白,其中 HSF1、2 和 4 均普遍表达,HSF3 则仅在鸟类细胞中表达。HSF1 在热、重金属和氧化剂的刺激下会诱导热休克基因转录,HSF2 则与精子发生和红系细胞发育有关。HSF3 和 HSF4 的功能与 HSF1 和 HSF2 重叠。失活的 HSF1 作为单体形式存在,位于细胞浆和细胞核,但不结合 DNA (1,2)。受到应急刺激时,HSF1 被磷酸化,形成同源三聚体,结合 DNA 并激活热休克基因转录 (1,2)。HSF1 活性分别通过 PLK1 在 Ser419 位点磷酸化和 CaMKII 在 Ser230 位点的磷酸化正调控,前者增强核转位,后者增强反式激活 (3,4)。或者,HSF1 的活性分别通过 GSK3 和 ERK1 在 303 和 307 位点的丝氨酸磷酸化抑制,从而结合 14-3-3 蛋白并在细胞浆中隔离 HSF1 (5,6)。此外,热休克反应减弱时,直接结合 Hsp70、HSP40/Hdj-1 和 HSF 结合蛋白 1 (HSBP1) 会抑制 HSF1 (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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