Bad pSer75 Human ELISA Kit (ab133984)

Overview

• Product nameBad pSer75 Human ELISA Kit
• Precision

  Intra-assay

  Sample	n	Mean	SD	CV%
  Overall	7			6%
• Tests
  1 x 96 well plate
• Sample type
  Cell culture extracts, Tissue Extracts, Cell Lysate
• Assay typeSandwich
• Sensitivity
  3 µg/ml
• Recovery

  Sample specific recovery

  Sample type	Average %	Range
  Cell culture extracts		3µg/ml - 200µg/ml

• Species reactivity
  Reacts with: Human, Monkey
  Predicted to work with: Mouse
• Product overview

  BAD (Q92934) is a BH3-only pro-apoptotic member of the Bcl-2 family. BAD competes for the binding to Bcl-X (L), Bcl-2 and Bcl-W, thereby affecting the level of heterodimerization of these proteins with BAX. BAD can reverse the death repressor activity of Bcl-X (L), but not that of Bcl-2. It appears to act as a link between growth factor receptor signaling and the apoptotic pathways. Bad is localized in mitochondrial outer membrane. Upon phosphorylation, Bad locates to the cytoplasm. Bad is expressed in a wide variety of tissues. 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. BAD is 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. The 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. The phosphorylation at Ser-99 by PKB/AKT1 is almost completely blocked by the apoptotic C-terminus cleavage product of PKN2 generated by caspases-3 activity during apoptosis.

• Tested applicationsSandwich ELISA more details

Properties

• Storage instructionsStore at +4°C. Please refer to protocols.

• Components	Identifier	1 x 96 tests
  ab133984 - 10X BAD pSer75 Detector Antibody		1 x 700µl
  10X HRP Label		1 x 1ml
  10X Wash Buffer	8209742	1 x 40ml
  ab62480 - BAD Microplate (12 x 8 antibody coated wells)		1 x 96 tests
  Extraction Buffer		1 x 15ml
  Incubation Buffer		1 x 60ml
  TMB Development Solution		1 x 12ml

• Research Areas
  • Kits/ Lysates/ Other
  • Kits
  • ELISA Kits
  • ELISA Kits
  • Phosphoprotein ELISA kits

Applications

Bad pSer75 Human ELISA Kit images

• 

  Sandwich ELISA - Bad pSer75 Human ELISA Kit (ab133984)
  Example positive control sample standard curve. A dilution series of extract in Incubation Buffer in the working range of the assay. The extract was prepared from COS7 cells serum-starved overnight and then treated with 200 nM Phorbol 12-myristate 13-acetate (PMA) for one hour.
• 

  Sandwich ELISA - Bad pSer75 Human ELISA Kit (ab133984)
  Example experimental analysis of drug treatment of HeLa cells. Cells were treated with Ser/Thr phosphatase inhibitor Calyculin A, Phorbol 12-myristate 13-acetate (PMA) or drug’s vehicle, as indicated. The Relative levels of BAD Phospho S75 in HeLa cells interpolated from standard curve of vehicle-treated HeLa cells are shown.
• 

  Sandwich ELISA - Bad pSer75 Human ELISA Kit (ab133984)
  Example experimental analysis of drug treatment of COS7 cells. Cells were treated with Ser/Thr phosphatase inhibitor Calyculin A, Phorbol 12-myristate 13-acetate (PMA) or drug’s vehicle, as indicated. Relative levels of BAD Phospho S75 in COS7 cells interpolated from standard curve of vehicle-treated COS7 cells are shown.
• 

  Sandwich ELISA - Bad pSer75 Human ELISA Kit (ab133984)
  The BAD Phospho S75 ELISA specifically measures the phosphorylated Serine. Extracts of COS7 cells (induced with PMA) were treated with increasing concentrations of λ protein phosphatase or left untreated (Contr), and relative BAD phospho Ser75 were determined using this kit. Dilutions of extracts of COS7 cells treated with PMA were used to construct the standard curve.
• 

  Western blot - Bad pSer75 Human ELISA Kit (ab133984)
  The detector antibody used in this kit specifically detects the phosphorylated BAD as determined by Western blotting. A and B: Extracts of COS7 cells (induced with PMA) were treated with increasing concentrations of λ protein phosphatase (lane 3, 400x diluted; lane 4, 100x diluted; lane 5, 25x diluted) or left untreated (lane 2). Samples were analyzed by Western blotting using the BAD Phospho S75 Detector Antibody (A) or total BAD protein antibody (B). C and D: Extracts of HeLa cells treated with vehicle (lane 2) or Calyculin (lane 3) and extracts of Cos7 cells treated with vehicle (lane 4) or PMA (lane 5) were analyzed by Western blotting using the BAD Phospho S75 Detector Antibody (C) or total BAD protein antibody (D). Note slower mobility of phosphorylated BAD in COS7 cells compared to dephosphorylated BAD.

Protocols

Protocol Booklet