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11865
Phospho-Bad (Ser112) (40A9) Rabbit mAb (PE Conjugate)
Antibody Conjugates

Phospho-Bad (Ser112) (40A9) Rabbit mAb (PE Conjugate) #11865

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APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous Rabbit IgG
Flow Cytometry
Flow Cytometry

Flow cytometric analysis of HCT 116 cells, treated with Staurosporine #9953 (1 uM, 4 hr; blue) or TPA #4174 (200 nM, 30 min; green), using Phospho-Bad (Ser112) (40A9) Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Application Dilutions
Flow Cytometry 1:50
Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibodies. Protect from light. Do not freeze.

Phospho-Bad (Ser112) (40A9) Rabbit mAb (PE Conjugate) detects endogenous levels of Bad only when phosphorylated at Ser112. The Ser112 nomenclature is based upon the mouse sequence. The analogous phosphorylation site is Ser75 in human and Ser113 in rat. This antibody does not detect Bad phosphorylated at other sites, nor does it detect related family members.

Species Reactivity:

Human, Mouse, Rat, Monkey

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser112 of mouse Bad.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in monkey cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Bad (Ser112) (40A9) Rabbit mAb #5284.

Bad is a proapoptotic member of the Bcl-2 family that promotes cell death by displacing Bax from binding to Bcl-2 and Bcl-xL (1,2). Survival factors, such as IL-3, inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (2). Phosphorylation at these sites promotes binding of Bad to 14-3-3 proteins to prevent an association between Bad with Bcl-2 and Bcl-xL (2). Akt phosphorylates Bad at Ser136 to promote cell survival (3,4). Bad is phosphorylated at Ser112 both in vivo and in vitro by p90RSK (5,6) and mitochondria-anchored PKA (7). Phosphorylation at Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL (8-10).

  1. Yang, E. et al. (1995) Cell 80, 285-91.
  2. Zha, J. et al. (1996) Cell 87, 619-28.
  3. Datta, S.R. et al. (1997) Cell 91, 231-41.
  4. del Peso, L. et al. (1997) Science 278, 687-9.
  5. Bonni, A. et al. (1999) Science 286, 1358-62.
  6. Tan, Y. et al. (1999) J Biol Chem 274, 34859-67.
  7. Harada, H. et al. (1999) Mol Cell 3, 413-22.
  8. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
  9. Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.
  10. Datta, S. et al. (2000) Mol. Cell 6, 41-51.
Entrez-Gene Id
572
Swiss-Prot Acc.
Q92934
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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11865S		 100 µl  (50 tests) $ 327.0

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