重组Anti-pro Caspase-3抗体[E61] (ab32150)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E61] to pro Caspase-3
- Suitable for: IP, WB, IHC-P, ICC/IF, Flow Cyt (Intra)
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
概述
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产品名称
Anti-pro Caspase-3抗体[E61]
参阅全部 pro Caspase-3 一抗 -
描述
兔单克隆抗体[E61] to pro Caspase-3 -
宿主
Rabbit -
特异性
The antibody only recognizes the pro-form of Caspase-3. It does not react with the cleaved forms (active enzyme) of Caspase-3. -
经测试应用
适用于: IP, WB, IHC-P, ICC/IF, Flow Cyt (Intra)more details -
种属反应性
与反应: Human -
免疫原
Synthetic peptide within Human pro Caspase-3 (N terminal). The exact sequence is proprietary.
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阳性对照
- WB: Hap1 & HeLa cells. ICC/IF: Jurkat cells. IHC-P: cervical carcinoma tissue. IP: HeLa whole cell lysate. Flow Cyt (Intra): Jurkat cells.
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常规说明
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
E61 -
同种型
IgG -
研究领域
相关产品
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Alternative Versions
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Assay kits
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Compatible Secondaries
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Isotype control
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab32150于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。
应用 | Ab评论 | 说明 |
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IP |
1/20.
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WB | (2) |
1/1000. Detects a band of approximately 35 kDa.
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IHC-P |
1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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ICC/IF |
1/200.
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Flow Cyt (Intra) |
1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
说明 |
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IP
1/20. |
WB
1/1000. Detects a band of approximately 35 kDa. |
IHC-P
1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
ICC/IF
1/200. |
Flow Cyt (Intra)
1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
靶标
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相关性
Caspases are a family of cysteine proteases that are key mediators of programmed cell death or apoptosis. The precursor form of all caspases is composed of a prodomain, and large and small catalytic subunits. The active forms of caspases are generated by several stimuli including ligand-receptor interactions, growth factor deprivation and inhibitors of cellular functions. All known caspases require cleavage adjacent to aspartates to liberate one large and one small subunit, which associate into a2b2 tetramer to form the active enzyme. Gene for Caspase 3 also known as Yama, CPP32, and apopain codes for a 32-kDa protein. Caspase 3 cleaves the death substrate poly(ADP-ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis and is inhibitable by the CrmA protein. Other Caspase 3 substrates include DNA-PK, actin, GAS2, and procaspase-6, etc. Caspase 3 is activated by cleavage events at Asp-28/Ser-29 (between N-terminal pro-domain) and Asp-175/Ser-176 (between large and small subunits) to generate a large subunit of 17-kDa and a small subunit of 12-kDa. -
细胞定位
Cytoplasmic -
数据库链接
- Entrez Gene: 836 Human
- Omim: 600636 Human
- SwissProt: P42574 Human
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别名
- CASP3 antibody
- Caspase 3 antibody
- Caspase 3 apoptosis related cysteine peptidase antibody
see all
图片
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All lanes : Anti-pro Caspase-3 antibody [E61] (ab32150) at 1/1000 dilution
Lane 1 : Wild-type HeLa Treated Staurosporine (2uM, 4h) cell lysate
Lane 2 : CASP3 knockout HeLa Treated Staurosporine (2uM, 4h) cell lysate
Lane 3 : Wild-type HeLa Vehicle Control Staurosporine (0uM, 4h) cell lysate
Lane 4 : CASP3 knockout HeLa Vehicle Control Staurosporine (0uM, 4h) cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 30 kDa why is the actual band size different from the predicted?Anti-CASP3 antibody [E61] (ab32150) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32150 was shown to bind specifically to CASP3. A band was observed at 30 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in CASP3 knockout cell line. To generate this image, wild-type and CASP3 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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Lane 1: Wild-type HAP1 cell lysate
Lane 2: Wild-type HAP1 cell lysate + Staurosporine (1μM for 4h)
Lane 3: Caspase-3 knockout HAP1 cell lysate
Lane 4: Caspase-3 knockout HAP1 cell lysate + Staurosporine (1μM for 4h)
Lanes 1 - 4: Merged signal (red and green). Green - ab32150 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32150 was shown to specifically react with pro Caspase 3 when Caspase 3 knockout samples were used. Wild-type and Caspase 3 knockout samples (± Staurosporine treatment) were subjected to SDS-PAGE. ab32150 and ab8245 (loading control to GAPDH) were diluted to 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging. -
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells labelling pro Caspase-3 with primary antibody anti-pro Caspase-3 (ab32150) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) secondary antibody at 1/1000 dilution. Confocal image showing cytoplasmic staining in Jurkat cells. Anti-alpha Tubulin antibody (DM1A) - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution. The nuclear counter stain is DAPI (blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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Immunohistochemical analysis of human paraffin-embedded cervical carcinoma tissue using ab32150 at 1/500 dilution.
Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Overlay histogram showing Jurkat cells stained with ab32150 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32150, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was anti-rabbit DyLight® 488 (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
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pro Caspase-3 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg with ab32150 at 1/20 dilution (0.6µg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg
Lane 2: ab32150 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab32150 in HeLa whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
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Anti-pro Caspase-3 antibody [E61] (ab32150) at 1/1000 dilution + Hela cell lysate
Observed band size: 35 kDa why is the actual band size different from the predicted?
实验方案
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
数据表及文件
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SDS download
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Datasheet download
文献 (132)
ab32150 被引用在 132 文献中.
- Zhao Q et al. The central role of a two-way positive feedback pathway in molecular targeted therapies-mediated pyroptosis in anaplastic thyroid cancer. Clin Transl Med 12:e727 (2022). PubMed: 35184413
- Li Y et al. microRNA-378a-3p regulates the progression of hepatocellular carcinoma by regulating PD-L1 and STAT3. Bioengineered 13:4730-4743 (2022). PubMed: 35184646
- Zhang W et al. Paired box 5 increases the chemosensitivity of esophageal squamous cell cancer cells by promoting p53 signaling activity. Chin Med J (Engl) 135:606-618 (2022). PubMed: 35191417
- Fan J et al. lncRNA LEF1-AS1 Acts as a Novel Biomarker and Promotes Hypopharyngeal Squamous Cell Carcinoma Progression and Metastasis by Targeting the miR-221-5p/GJA1 Axis. Dis Markers 2022:3881310 (2022). PubMed: 35371339
- Luo H et al. MAT2A facilitates PDCD6 methylation and promotes cell growth under glucose deprivation in cervical cancer. Cell Death Discov 8:176 (2022). PubMed: 35396512