Recombinant Anti-Bak antibody [Y164] (ab32371)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y164] to Bak
- Suitable for: IP, Flow Cyt (Intra), WB, IHC-P
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-Bak antibody [Y164]
See all Bak primary antibodies -
Description
Rabbit monoclonal [Y164] to Bak -
Host species
Rabbit -
Specificity
This antibody recognises Bak. The antibody does not cross-react with other Bcl2 members.
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Tested applications
Suitable for: IP, Flow Cyt (Intra), WB, IHC-Pmore details
Unsuitable for: ICC/IF -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Recombinant Human Bak protein (ab114337), HAP1, HeLa and HEK-293 cell lysates; human heart and fetal heart lysates. IHC-P: Human pancreatic carcinoma, stomach carcinoma and normal stomach tissue. IP: HCT 116 and HeLa cell lysates. Flow Cyt (intra): HeLa cells.
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General notes
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.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.21% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
Y164 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32371 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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IP | (1) |
Use at an assay dependent concentration.
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Flow Cyt (Intra) |
1/10 - 1/20.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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WB | (1) |
1/10000. Predicted molecular weight: 23 kDa.
For unpurified use at 1/1000 - 1/5000. |
IHC-P | (1) |
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Notes |
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IP
Use at an assay dependent concentration. |
Flow Cyt (Intra)
1/10 - 1/20. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
1/10000. Predicted molecular weight: 23 kDa. For unpurified use at 1/1000 - 1/5000. |
IHC-P
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Target
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Function
In the presence of an appropriate stimulus, accelerates programmed cell death by binding to, and antagonizing the anti-apoptotic action of BCL2 or its adenovirus homolog E1B 19k protein. Low micromolar levels of zinc ions inhibit the promotion of apoptosis. -
Tissue specificity
Expressed in a wide variety of tissues, with highest levels in the heart and skeletal muscle. -
Sequence similarities
Belongs to the Bcl-2 family. -
Domain
Intact BH3 motif is required by BIK, BID, BAK, BAD and BAX for their pro-apoptotic activity and for their interaction with anti-apoptotic members of the Bcl-2 family. -
Cellular localization
Mitochondrion membrane. - Information by UniProt
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Database links
- Entrez Gene: 578 Human
- Omim: 600516 Human
- SwissProt: Q16611 Human
- Unigene: 485139 Human
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Alternative names
- Apoptosis regulator BAK antibody
- BAK antibody
- BAK like antibody
see all
Images
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Immunohistochemical analysis of paraffin-embedded fixed (A) Wild-type HeLa (human cervix adenocarcinoma epithelial cell) cell pellet. (B) BAK1 knockout HeLa (ab265277) cell pellet staining Bak with ab32371 at 1/2000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Counter-staining used was hematoxylin.
Positive staining on (A) Wild-type HeLa cell pellet, no staining on (B) BAK1 knockout HeLa (ab265277) cell pellet.
The section was incubated with ab32371 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrumentHeat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.
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Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized BAK1 KO Hela (human BAK1 knockout cervical adenocarcinoma epithelial cell, Left) /Parental Hela (Right) cells labelling Bak with ab32371 at 1/500 dilution (0.1 µg)(Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody. -
All lanes : Anti-Bak antibody [Y164] (ab32371) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BAK1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 23 kDaLanes 1- 2: Merged signal (red and green). Green - ab32371 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab32371 was shown to react with Bak in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265277 (knockout cell lysate ab257077) was used. Wild-type HeLa and BAK1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32371 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human pancreatic carcinoma tissue sections labeling Bak with Purified ab32371 at 1:200 dilution (2.98 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control. -
All lanes : Anti-Bak antibody [Y164] (ab32371)
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : BAK knockout HAP1 whole cell lysate
Lane 3 : Human Heart whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 23 kDaLanes 1 - 3: Merged signal (red and green). Green - ab32371 observed at 25 kDa. Red - loading control, ab9484, observed at 37 kDa.
Unpurified ab32371 was shown to specifically recognize BAK in wild-type HAP1 cells. No band was observed when BAK knockout cells were examined. Wild-type and BAK knockout samples were subjected to SDS-PAGE. Unpurified ab32371 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.
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ab32371 (purified) at 1:20 dilution (2μg) immunoprecipitating Bak in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+): ab32371 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32371 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human stomach tissue sections labeling Bak with Purified ab32371 at 1:200 dilution (2.98 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control. -
All lanes : Anti-Bak antibody [Y164] (ab32371) at 1/10000 dilution (purified)
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 3 : Human fetal heart lysates
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution
Predicted band size: 23 kDa
Observed band size: 23 kDaBlocking and diluting buffer: 5% NFDM/TBST
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Anti-Bak antibody [Y164] (ab32371) at 1/5000 dilution (unpurified) + HeLa cell lysate
Predicted band size: 23 kDa
Observed band size: 23 kDa -
Immunohistochemical analysis of Bak expression in paraffin embedded human stomach carcinoma, using 1/250 unpurified ab32371.
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Bak was immunoprecipitated from HCT116 p53-/- cell line whole cell lysate with unpurified ab32371 at 1/100 dilution.
Western blot was performed from the immunoprecipitate using ab32371 at 1/2000 dilution.
Datasheets and documents
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SDS download
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Datasheet download
References (57)
ab32371 has been referenced in 57 publications.
- Yang Y et al. SARS-CoV-2 membrane protein causes the mitochondrial apoptosis and pulmonary edema via targeting BOK. Cell Death Differ 29:1395-1408 (2022). PubMed: 35022571
- Zhou P et al. SHP2 Inhibitors Show Anti-Myeloma Activity and Synergize With Bortezomib in the Treatment of Multiple Myeloma. Front Pharmacol 13:841308 (2022). PubMed: 35462913
- He L et al. miRNA-138-5p suppresses cigarette smoke-induced apoptosis in testicular cells by targeting Caspase-3 through the Bcl-2 signaling pathway. J Biochem Mol Toxicol 35:e22783 (2021). PubMed: 33856081
- Surman DR et al. Therapeutic Synergy in Esophageal Cancer and Mesothelioma Is Predicted by Dynamic BH3 Profiling. Mol Cancer Ther 20:1469-1480 (2021). PubMed: 34088830
- Jiang L et al. Anticancer effects of dihydromyricetin on the proliferation, migration, apoptosis and in vivo tumorigenicity of human hepatocellular carcinoma Hep3B cells. BMC Complement Med Ther 21:194 (2021). PubMed: 34229692