Recombinant Anti-Bad antibody [Y208] (ab32445)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y208] to Bad
- Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-Bad antibody [Y208]
See all Bad primary antibodies -
Description
Rabbit monoclonal [Y208] to Bad -
Host species
Rabbit -
Specificity
This antibody does not cross-react with other Bcl-2 members. The mouse and rat recommendation is based on the IHC-P results. We do not guarantee WB for mouse and rat.
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Tested applications
Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IFmore details -
Species reactivity
Reacts with: Mouse, Rat, Human
Predicted to work with: Dog -
Immunogen
Synthetic peptide within Human Bad (N terminal). The exact sequence is proprietary.
(Peptide available asab206866) -
Positive control
- ICC/IF: HeLa cells; IHC-P: Human ovarian cancer, Human, Mouse and Rat kidney tissue; Flow Cyt (intra): MCF7 cells. WB: HeLA and HepG2 whole cell lysate.
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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.
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), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
Y208 -
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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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32445 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) |
1/20 - 1/50.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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WB | (7) |
1/1000. Detects a band of approximately 23 kDa (predicted molecular weight: 18 kDa).
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IHC-P | (1) |
1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
The mouse and rat recommendation is based on the IHC-P results. We do not guarantee WB for mouse and rat." in the "Specificity" section and "WB Application |
ICC/IF | (1) |
1/50.
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Notes |
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Flow Cyt (Intra)
1/20 - 1/50. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
1/1000. Detects a band of approximately 23 kDa (predicted molecular weight: 18 kDa). |
IHC-P
1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. The mouse and rat recommendation is based on the IHC-P results. We do not guarantee WB for mouse and rat." in the "Specificity" section and "WB Application |
ICC/IF
1/50. |
Target
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Function
Promotes cell death. Successfully competes for the binding to Bcl-X(L), Bcl-2 and Bcl-W, thereby affecting the level of heterodimerization of these proteins with BAX. Can reverse the death repressor activity of Bcl-X(L), but not that of Bcl-2 (By similarity). Appears to act as a link between growth factor receptor signaling and the apoptotic pathways. -
Tissue specificity
Expressed in a wide variety of tissues. -
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. -
Post-translational
modificationsPhosphorylated on one or more of Ser-75, Ser-99, Ser-118 and Ser-134 in response to survival stimuli, which blocks its pro-apoptotic activity. Phosphorylation on Ser-99 or Ser-75 promotes heterodimerization with 14-3-3 proteins. This interaction then facilitates the phosphorylation at Ser-118, a site within the BH3 motif, leading to the release of Bcl-X(L) and the promotion of cell survival. Ser-99 is the major site of AKT/PKB phosphorylation, Ser-118 the major site of protein kinase A (CAPK) phosphorylation. Ser-75 is phosphorylated by AKT/PKB, protein kinase A and PIM2. -
Cellular localization
Mitochondrion outer membrane. Cytoplasm. Upon phosphorylation, locates to the cytoplasm. - Information by UniProt
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Database links
- Entrez Gene: 483763 Dog
- Entrez Gene: 572 Human
- Entrez Gene: 12015 Mouse
- Entrez Gene: 64639 Rat
- Omim: 603167 Human
- SwissProt: Q92934 Human
- SwissProt: Q61337 Mouse
- SwissProt: O35147 Rat
see all -
Alternative names
- AI325008 antibody
- BAD antibody
- BAD_HUMAN antibody
see all
Images
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All lanes : Anti-Bad antibody [Y208] (ab32445) at 1/2000 dilution (Purified)
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 18 kDa -
All lanes : Anti-Bad antibody [Y208] (ab32445) at 1/2000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BAD knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 18 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab32445 was shown to react with Bad in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264843 (knockout cell lysate ab256847) was used. Wild-type HeLa and BAD 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. ab32445 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 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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All lanes : Anti-Bad antibody [Y208] (ab32445) at 1/2000 dilution
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : BAD knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MCF7 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 18 kDaLanes 1 - 4: Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab32445 was shown to specifically recognise BAD in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when BAD knockout cells were examined. Wild-type and BAD knockout samples were subjected to SDS-PAGE. Ab32445 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 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 ovarian cancer tissue sections labeling Bad with purified ab32445 at 1/1000 dilution (0.14 µg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. -
Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Bad with purified ab32445 at 1/50 dilution (2.9 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling Bad with purified ab32445 at 1/1000 dilution (0.14 µg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling Bad with purified ab32445 at 1/1000 dilution (0.14 µg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling Bad with purified ab32445 at 1/1000 dilution (0.14 µg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. -
Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labelling Bad with purified ab32445 at 1/20 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).
Protocols
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.
Datasheets and documents
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SDS download
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Datasheet download
References (121)
ab32445 has been referenced in 121 publications.
- Wang L et al. Dexmedetomidine prevents cardiomyocytes from hypoxia/reoxygenation injury via modulating tetmethylcytosine dioxygenase 1-mediated DNA demethylation of Sirtuin1. Bioengineered 13:9369-9386 (2022). PubMed: 35387565
- Ma Z et al. ZMAT1 acts as a tumor suppressor in pancreatic ductal adenocarcinoma by inducing SIRT3/p53 signaling pathway. J Exp Clin Cancer Res 41:130 (2022). PubMed: 35392973
- Cai J et al. SERP1 reduces inchoate acute hepatic injury through regulation of endoplasmic reticulum stress via the GSK3β/β‑catenin/TCF/LEF signaling pathway. Mol Med Rep 25:N/A (2022). PubMed: 35419615
- Huang X et al. La protein regulates protein expression by binding with the mRNAs of target genes and participates the pathological process of ovarian cancer. Front Oncol 12:763480 (2022). PubMed: 36110943
- Yan S et al. Expression of PD-L1 and YWHAZ in Patients with Diffuse Large B Cell Lymphoma: A Possible Association with the Prognosis of Lymphoma. J Immunol Res 2022:5633096 (2022). PubMed: 36213322