Validated using a knockout cell line
Recombinant
RabMAb

Anti-Bad antibody [Y208] (ab32445)

Overview

  • Product name
    Anti-Bad antibody [Y208]
    See all Bad primary antibodies
  • Description
    Rabbit monoclonal [Y208] to Bad
  • Specificity
    The antibody does not cross-react with other Bcl-2 members.
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Dog, Human
  • Immunogen

    A synthetic peptide corresponding to residues in the N-term of human Bad.

  • Positive control
    • Hela cell lysate, human lymphoma.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

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
WB 1/2000 - 1/5000. Detects a band of approximately 23 kDa (predicted molecular weight: 18 kDa).
IHC-P Use at an assay dependent concentration.
ICC/IF 1/250 - 1/500.
Flow Cyt 1/20 - 1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP 1/100.

Target

  • 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
    modifications
    Phosphorylated 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
  • Database links
  • Alternative names
    • AI325008 antibody
    • BAD antibody
    • BAD_HUMAN antibody
    • BBC 2 antibody
    • BBC2 antibody
    • BBC6 antibody
    • Bcl 2 Antagonist of Cell Death antibody
    • Bcl 2 Binding Component 6 antibody
    • BCL X / BCL 2 Binding Protein antibody
    • BCL X Binding Protein antibody
    • Bcl XL/Bcl 2 Associated Death Promoter antibody
    • Bcl-2-binding component 6 antibody
    • Bcl-2-like protein 8 antibody
    • Bcl-XL/Bcl-2-associated death promoter antibody
    • Bcl2 antagonist of cell death antibody
    • BCL2 antagonist of cell death protein antibody
    • BCL2 associated agonist of cell death antibody
    • Bcl2 Associated Death Promoter antibody
    • BCL2 binding component 6 antibody
    • BCL2 binding protein antibody
    • Bcl2 Like 8 Protein antibody
    • Bcl2-L-8 antibody
    • BCL2L8 antibody
    • Proapoptotic BH3 Only Protein antibody
    see all

Images



  • Predicted band size : 18 kDa

    Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
    Lane 2: BAD knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)
    Lane 4: MCF7 whole cell lysate (20 µg)

    Lanes 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.

  • Immunocytochemical analysis of 2% paraformaldehyde-fixed,0.1% Triton-X100 permeabilized RAW 264.7 labeling Bad with ab32445 at 1/250dilution,followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 568) secondary antibody at 1/1000 dilution.

  • Immunohistochemical staining of paraffin-embedded human lymphoma using ab32445 at 1/100 dilution.
  • Anti-Bad antibody [Y208] (ab32445) at 1/5000 dilution + Hela cell lysate

    Predicted band size : 18 kDa
    Observed band size : 23 kDa (why is the actual band size different from the predicted?)
  • Overlay histogram showing MCF-7 cells stained with ab32445 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab32445, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in MCF-7 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

References

This product has been referenced in:
  • Beffagna G  et al. Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index. PLoS One 12:e0169454 (2017). Read more (PubMed: 28081183) »
  • Liu Y  et al. Effects of bufalin on the mTOR/p70S6K pathway and apoptosis in esophageal squamous cell carcinoma in nude mice. Int J Mol Med 40:357-366 (2017). Read more (PubMed: 28656204) »

See all 20 Publications for this product

Customer reviews and Q&As

ab33906 lot YH090207C - 0.3260 mg/ml

ab33862 lot YH082410D - 0.6380 mg/ml

ab32429 lot YG080503 - 0.0790 mg/ml

ab40660 lot YI070802CS - 0.1030 mg/ml

ab32445 lot YI072804DS - 0.0250 mg/ml

Application
Immunocytochemistry
Sample
Mouse Cultured Cells (RAW 264.7)
Specification
RAW 264.7
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton-X100 in 2% BSA for 15min
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
Username

Dr. Mahesh Shivananjappa

Verified customer

Submitted May 19 2012

Thank you for contacting us.
There is a wide range of secondaries suitable to detect either ab32445 or ab33862 in Flow Cytometry. I have conducted a search through our Secondaries search tool in the Abcam Website and found more than 100 matches, d...

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Thank you for contacting Abcam.

Included in this email are the instructions for returning your item to Abcam. When you return the goods, please ensure that you follow the procedures as below:

- Return in the original Abcam box with...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Glioblastoma neurospheres cells (primary))
Loading amount
30 µg
Specification
Glioblastoma neurospheres cells (primary)
Gel Running Conditions
Reduced Denaturing (12.5% gel)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Dec 06 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Platelets)
Loading amount
20 µg
Specification
Platelets
Treatment
Calcium Ionophore for 1 h
Gel Running Conditions
Reduced Denaturing (4-20%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Dr. Mahesh Shivananjappa

Verified customer

Submitted Nov 14 2011

Thank you for contacting us. I can confirm that this antibody does recongize the phosphorylated and unphosphorylated form of BAD. Indeed, there is no phosphorylation site on the immunogen sequences used for this antibody. I hope this inf...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Flow Cytometry
Sample
Human Cell (Mononuclear Cell Preparations)
Specification
Mononuclear Cell Preparations
Fixation
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton-X100 in 2% BSA for 15min
Gating Strategy
Monocytes Lymphocytes
Username

Dr. Mahesh Shivananjappa

Verified customer

Submitted Mar 30 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (PMN)
Specification
PMN
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton-X100 in 2% BSA for 30min
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Dec 20 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (293T)
Total protein in input
200 µg
Specification
293T
Immuno-precipitation step
Protein A
Username

Abcam user community

Verified customer

Submitted Dec 31 2007

1-10 of 13 Abreviews or Q&A

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