重组Anti-Caspase-3抗体[E87] (ab32351)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E87] to Caspase-3
- Suitable for: ICC/IF, WB, IHC-P, Flow Cyt, IP
- Knockout validated
- Reacts with: Human
概述
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产品名称
Anti-Caspase-3抗体[E87]
参阅全部 Caspase-3 一抗 -
描述
兔单克隆抗体[E87] to Caspase-3 -
宿主
Rabbit -
特异性
This antibody is specific for the pro form and the p17 cleaved form of human Caspase-3. -
Tested Applications & Species
Application Species Flow Cyt HumanICC/IF HumanIHC-P HumanIP HumanWB Human -
免疫原
Synthetic peptide within Human Caspase-3 aa 50-150. The exact sequence is proprietary.
Database link: P42574 -
阳性对照
- WB: Jurkat whole cell lysate (ab7899); Wild-type HAP1 whole cell lysate; Ramos and HEK-293 cell lysates. IHC-P: Human tonsil and cervical carcinoma tissue. ICC/IF: Jurkat cells. Flow Cyt: HeLa and Ramos cells. IP: HeLa whole cell lysate.
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常规说明
Rat reactivity: We have preliminary internal testing data indicating this antibody does not react with mouse caspase 3. Internal testing and customer reports also indicate non-reactivity with rat capsase 3, contrary to earlier positive reports. Please contact us for more information.
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.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.
One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.
Learn more here.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
E87 -
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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Conjugation kits
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
应用
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32351 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Tested applications are guaranteed to work and covered by our Abpromise guarantee.
Predicted to work for this combination of applications and species but not guaranteed.
Does not work for this combination of applications and species.
应用 | Species |
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Flow Cyt |
Human
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ICC/IF |
Human
|
IHC-P |
Human
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IP |
Human
|
WB |
Human
|
应用 | Ab评论 | 说明 |
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ICC/IF | (3) |
1/500.
For unpurified, use 1/25. |
WB | (7) |
1/5000. Detects a band of approximately 35 kDa (predicted molecular weight: 32 kDa).
|
IHC-P | (2) |
1/25 - 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
|
Flow Cyt | (1) |
1/180 - 1/1000.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
IP |
1/10 - 1/50.
|
说明 |
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ICC/IF
1/500. For unpurified, use 1/25. |
WB
1/5000. Detects a band of approximately 35 kDa (predicted molecular weight: 32 kDa). |
IHC-P
1/25 - 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. |
Flow Cyt
1/180 - 1/1000. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
IP
1/10 - 1/50. |
靶标
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功能
Involved in the activation cascade of caspases responsible for apoptosis execution. At the onset of apoptosis it proteolytically cleaves poly(ADP-ribose) polymerase (PARP) at a '216-Asp-
-Gly-217' bond. Cleaves and activates sterol regulatory element binding proteins (SREBPs) between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. Cleaves and activates caspase-6, -7 and -9. Involved in the cleavage of huntingtin. -
组织特异性
Highly expressed in lung, spleen, heart, liver and kidney. Moderate levels in brain and skeletal muscle, and low in testis. Also found in many cell lines, highest expression in cells of the immune system. -
序列相似性
Belongs to the peptidase C14A family. -
翻译后修饰
Cleavage by granzyme B, caspase-6, caspase-8 and caspase-10 generates the two active subunits. Additional processing of the propeptides is likely due to the autocatalytic activity of the activated protease. Active heterodimers between the small subunit of caspase-7 protease and the large subunit of caspase-3 also occur and vice versa.
S-nitrosylated on its catalytic site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway, associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active site thiol. -
细胞定位
Cytoplasm. - Information by UniProt
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数据库链接
- Entrez Gene: 836 Human
- Omim: 600636 Human
- SwissProt: P42574 Human
- Unigene: 141125 Human
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别名
- A830040C14Rik antibody
- Apopain antibody
- CASP 3 antibody
see all
图片
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All lanes : Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution
Lane 1 : DMSO control wild-type HAP1 whole cell lysate
Lane 2 : Staurosporine treated wild-type HAP1 whole cell lysate
Lane 3 : DMSO control CASP3 knockout HAP1 whole cell lysate
Lane 4 : Staurosporine treated CASP3 knockout HAP1 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 32 kDaLanes 1 - 4: Merged signal (red and green). Green - ab32351 observed at 31 kDa. Red - loading control, ab130007, observed at 130 kDa.
ab32351 was shown to recognize Caspase 3 in wild-type HAP1 cells as signal was lost at the expected MW in HAP1 Staurosporine Treated (CASP3) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HAP1 Staurosporine Treated (CASP3) knockout samples were subjected to SDS-PAGE. ab32351 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [E87] (ab32351)Immunohistochemical staining of paraffin embedded human tonsil with purified ab32351 at a working dilution of 1/100. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
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Immunofluorescence staining of Jurkat (human T cell leukemia cell line from peripheral blood) cells with purified ab32351 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab32351 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [E87] (ab32351)
Unpurified ab32351, at a 1/25 dilution, staining Capase-3 in paraffin embedded human cervical carcinoma tissue by Immunohistochemistry.
Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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All lanes : Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution (purified)
Lane 1 : untreated Jurkat (human T cell leukemia cell line from peripheral blood)cell lysate
Lane 2 : Jurkat treated with staurosporine
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution
Predicted band size: 32 kDa
Observed band size: 17,35 kDa why is the actual band size different from the predicted?Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST -
All lanes : Anti-Caspase-3 antibody [E87] (ab32351) at 1/5000 dilution (purified)
Lane 1 : Ramos (human Burkitt's lymphoma cell line) cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution
Predicted band size: 32 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST -
ab32351 (purified) at 1/50 immunoprecipitating Cullin 1 in 10 μg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate (Lanes 1 and 2, observed at 35 kDa). Lane 3 - PBS. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection (1/1500). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST
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Overlay histogram showing Ramos (human Burkitt's lymphoma cell line) cells fixed in 4% PFA and stained with purified ab32351 at a dilution of 1 in 180 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).
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Carried out with unpurified antibody. Lane 1 = Caspase 3 protein (Active) (ab52314) 20 ng. Lane 2 = Caspase 9 protein (Active) (ab52203) 20 ng. Lane 3 = Extract of HeLa (human epithelial cell line from cervix adenocarcinoma) cells treated with vehicle (ab136806) 20 ug. Lane 4 = Extract of HeLa cells treated with staurosporine (ab136806) 20 ug. SDS PAGE performed under reducing conditions (100 mM DTT Sample heated at 50°C). Primary : Lanes 1-4: Anti Caspase 3 antibody (ab32351) at 1:1000 dilution. Secondary : Lanes 1-4: Goat anti rabbit IgG(H&L)-HRP at 1:10000. Development: ECL for 10 min exposure. Blocking: in 5% Milk + PBS overnight at 4 C. Primary antibody: in 5% Milk + PBS for 2 hours at RT. Secondary antibody: in 5% Milk + PBS for 2 hours at RT. Predicted band size : 32 kDa and 17 kDa. Observed band size : 32 kDa and 17 kDa.
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Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with unpurfied ab32351 (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 followed by the antibody (ab32351, 1/1000 dilution) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.
Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
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 (130)
ab32351 被引用在 130 文献中.
- Bai S et al. Comparative analysis of pathophysiological parameters between emphysematous smokers and emphysematous patients with COPD. Sci Rep 10:420 (2020). PubMed: 31942006
- Shi Y MEG3 regulates apoptosis of adipose-derived stem cells. Mol Med Rep 21:2435-2442 (2020). PubMed: 32323784
- Xie Y et al. SNHG7 Facilitates Hepatocellular Carcinoma Occurrence by Sequestering miR-9-5p to Upregulate CNNM1 Expression. Cancer Biother Radiopharm N/A:N/A (2020). PubMed: 32397799
- Tang C et al. Antagonizing effect of icaritin on apoptosis and injury of hippocampal neurocytes induced by amyloid beta via GR/BDNF signaling pathway. J Recept Signal Transduct Res N/A:1-10 (2020). PubMed: 32476534
- Jiang W et al. Inhibiting PKCß2 protects HK-2 cells against meglumine diatrizoate and AGEs-induced apoptosis and autophagy. Ann Transl Med 8:293 (2020). PubMed: 32355737