Recombinant Anti-Bak antibody [Y164] - BSA and Azide free (ab220790)
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 - BSA and Azide free
- Suitable for: Flow Cyt (Intra), WB, IHC-P, IP
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-Bak antibody [Y164] - BSA and Azide free
See all Bak primary antibodies -
Description
Rabbit monoclonal [Y164] to Bak - BSA and Azide free -
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: Flow Cyt (Intra), WB, IHC-P, IPmore 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: HeLa, Hap1, human heart cells. IP: HeLa and HCT116 cells. Flow Cyt (intra): HeLa cells. IHC-P: Human pancreatic carcinoma, stomach carcinoma and stomach tissue.
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General notes
ab220790 is the carrier-free version of ab32371.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
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. Do Not Freeze. -
Storage buffer
pH: 7.20
Constituent: PBS -
Carrier free
Yes -
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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Conjugation kits
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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 ab220790 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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Flow Cyt (Intra) |
Use at an assay dependent concentration.
ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 23 kDa.
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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IP |
Use at an assay dependent concentration.
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Notes |
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Flow Cyt (Intra)
Use at an assay dependent concentration. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
Use at an assay dependent concentration. Predicted molecular weight: 23 kDa. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
IP
Use at an assay dependent concentration. |
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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This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32371).
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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This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32371).
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 kDaThis data was developed using the same antibody clone in a different buffer formulation (ab32371).
Lanes 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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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.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32371).
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All lanes : Anti-Bak antibody [Y164] (ab32371) at 1/1000 dilution
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 kDaThis WB data was generated using the same anti-Bak antibody clone, Y164, in a different buffer formulation (cat# ab32371).
Lanes 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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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.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32371).
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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.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32371).
Protocols
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (21)
ab220790 has been referenced in 21 publications.
- Lauf PK et al. Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak. Am J Physiol Cell Physiol 308:C51-60 (2015). Human . PubMed: 25318106
- Holloway A et al. Resistance to UV-induced apoptosis by ß-HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase. Int J Cancer 136:2831-43 (2015). PubMed: 25408501
- Xiao Y et al. MCL-1 Is a Key Determinant of Breast Cancer Cell Survival: Validation of MCL-1 Dependency Utilizing a Highly Selective Small Molecule Inhibitor. Mol Cancer Ther 14:1837-47 (2015). WB ; Human . PubMed: 26013319
- Hearn JM et al. Potent organo-osmium compound shifts metabolism in epithelial ovarian cancer cells. Proc Natl Acad Sci U S A 112:E3800-5 (2015). Human . PubMed: 26162681
- Wang B et al. Role of Ku70 in deubiquitination of Mcl-1 and suppression of apoptosis. Cell Death Differ 21:1160-9 (2014). PubMed: 24769731