BAD pSer112 + Total PhosphoTracer ELISA Kit (ab119655)

Overview

  • Product nameBAD pSer112 + Total PhosphoTracer ELISA Kit
  • Detection methodFluorescent
  • Tests
    1 x 96 well plate
  • Sample type
    Cell culture extracts
  • Assay typeSemi-quantitative
  • Assay time
    2h 0m
  • Assay durationOne step assay
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Product overview

    PhosphoTracer assays use a traditional immuno-sandwich format, but with a major difference both the analyte and the assay reagents are added to the PhosphoTracer assay microplate at the same time. After a short incubation period, unbound assay reagents and analytes are washed away, and immuno-complexes containing both antibodies are detected. The process can take as little as 60 minutes to complete.

    PhosphoTracer kits also allow a higher degree of assay flexibility. In contrast to other ELISA formats, no antibodies are present on the assay microplate itself, so assays for several different targets can be performed in different wells on the same microplate. Simply mix the lysate with your target reagents of choice, using the microplate configuration of your choice.

    A whole new way of performing cellular assays, PhosphoTracer takes the hard work out of running a standard ELISA, while still giving the high quality results expected from a sandwich immunoassay. Fully self-contained kits are supplied in convenient 96-well packs. Simple to use and highly sensitive PhosphoTracer kits are designed to get results, fast.

     

    Abcam’s PhosphoTracer BAD assay kits are for detection of endogenous and transfected levels of BAD (GenBank Accession NP_004313) in cellular lysates. Phospho-BAD assays only detect BAD when phosphorylated at Ser112. Total BAD assays detect BAD irrespective of phosphorylation status.



    The substrate used with the HRP conjugated detection antibody is a combination of 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) (wavelength exc/em = 530-540nm / 590-600nm), a highly sensitive and stable substrate for HRP) and ADHP Dilution Buffer (a stabilized H2O2 solution). Learn more about the fluorogenic substrate, ADHP.

  • Notes

    Sensitivity: Phospho-BAD: 5,000 cells/well (Tested in PC3 cells), BAD: 2,500 cells/well (Tested in PC3 cells).

    Range: Phospho-BAD: 5,000-80,000 cells/well tested (Tested in PC3 cells), BAD: 2,500-80,000 cells/well tested (Tested in PC3 cells).

  • Tested applicationsSandwich ELISA more details
  • PlatformMicroplate

Properties

  • FunctionPromotes 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 specificityExpressed in a wide variety of tissues.
  • Sequence similaritiesBelongs to the Bcl-2 family.
  • DomainIntact 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 localizationMitochondrion outer membrane. Cytoplasm. Upon phosphorylation, locates to the cytoplasm.
  • Target information above from: UniProt accession Q92934 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names
    • BAD
    • BAD_HUMAN
    • BBC2
    • BCL X/BCL 2 binding protein
    • Bcl-2-binding component 6
    • Bcl-2-like protein 8
    • Bcl-XL/Bcl-2-associated death promoter
    • Bcl2 antagonist of cell death
    • BCL2 antagonist of cell death protein
    • BCL2 associated agonist of cell death
    • BCL2 binding component 6
    • BCL2 binding protein
    • Bcl2-L-8
    • BCL2L8
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab119655 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
Sandwich ELISA Use at an assay dependent concentration. Phospho-BAD (S112): Tested in PC3; BAD: Tested in PC3, MCF7, HEK293, HeLa, NIH3T3.

BAD pSer112 + Total PhosphoTracer ELISA Kit images

  • Various cell lines were seeded at 40K/mL in a 96 well plate in medium containing 10% FBS, and cultured overnight. The following day, cells were lysed with 120 µL/well of freshly prepared PhosphoTracer Lysis Mix with shaking for 10 mins at room temperature. 50 µL of lysate was transferred to replicate wells of a PhosphoTracer assay plate, and Antibody Mix specific for total BAD (50 µL/well) was added to the lysates. The plates were incubated for 1 hr at room temp, with shaking. Plates were washed and Substrate Mix was added. The plates were covered in foil and incubated for 10 min with shaking. Signal in the wells was determined using a plate reader. The results show BAD is detectable in several cell lines.
  • PC3 cells were seeded at 40K cells/well in medium containing 10% FBS in a 96 well microplate, and cultured overnight. The following day the culture medium was removed, and the cells were treated with various concentrations of UCN-01 diluted in serum-free medium for 60 mins. The medium was removed from the wells, and the cells were lysed with 120 µL/well Lysis Mix, with shaking for 10 min. 50 µL of lysate was transferred to replicate wells of a PhosphoTracer assay plate, and 50 µL/well Antibody Mix specific for either (A) phospho BAD or (B) total BAD was added to the lysates. The plates were incubated for 1 hr at room temp, with shaking. Plates were washed and Substrate Mix was added. The plates were covered in foil and incubated for 10 min with shaking. Signal in the wells was determined using a plate reader.

Protocols

References for BAD pSer112 + Total PhosphoTracer ELISA Kit (ab119655)

ab119655 has not yet been referenced specifically in any publications.

Product Wall

The surface of the plate is already coated and blocked, so adding another coating antibody will not help, and potentially harm the assay performance.

As a general rule, the antibodies that are added to the plate during the binding phase are b...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"