Revision 1

#9105Store at -20C

1 Kit

(4 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-Bad (Ser112) (40A9) Rabbit mAb 5284 20 µl 23 kDa Rabbit IgG
Phospho-Bad (Ser136) (D25H8) Rabbit mAb 4366 20 µl 23 kDa Rabbit IgG
Phospho-Bad (Ser155) Antibody 9297 20 µl 23 kDa Rabbit 
Bad (D24A9) Rabbit mAb 9239 20 µl 23 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Phospho-Bad Antibody Sampler Kit provides an economical means to investigate the role of Bad protein in apoptosis. The kit contains primary and secondary antibodies to perform two Western blots with each antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

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, 285-291.
  2. Zha, J. et al. (1996) Cell 87, 619-628.
  3. Datta, S.R. et al. (1997) Cell 91, 231-241.
  4. Peso, L. et al. (1997) Science 278, 687-689.
  5. Bonni, A. et al. (1999) Science 286, 1358-1362.
  6. Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.
  7. Harada, H. et al. (1999) Mol. Cell 3, 413-422.
  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.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    使用に関する制限

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    Revision 1
    #9105

    Phospho-Bad Antibody Sampler Kit

    Phospho-Bad Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from serum-starved COS cells treated with TPA (200 nM, 20 min) using Phospho-Bad (Ser112) (40A9) Rabbit mAb #5284. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Phospho-Bad Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from OVCAR8 cells using Bad (D24A9) Rabbit mAb #9239. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Phospho-Bad Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from ACHN cells, untreated or treated with Human Epidermal Growth Factor #8916 (100 ng/ml, 30 min), and C2C12 cells, untreated or insulin-treated (100 nM, 30 min), using Phospho-Bad (Ser136) (D25H8) Rabbit mAb (upper) or Bad (D24A9) Rabbit mAb #9239 (lower).
    Phospho-Bad Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from COS cells, untreated or TPA-treated, using Phospho-Bad (Ser112) (40A9) Rabbit mAb (upper) or Bad Antibody #9292 (lower).
    Phospho-Bad Antibody Sampler Kit: Image 5 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Phospho-Bad Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using Bad (D24A9) Rabbit mAb.
    Phospho-Bad Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of cell extracts from 293 cells transfected with Wild-type Bad, Bad (S112A/S136A), Bad (S155A) and treated with forskolin (30 µM), using Phospho-Bad (Ser155) Antibody (upper) and Bad Antibody #9292 (lower).
    Phospho-Bad Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from COS-7 cells, treated with Human Epidermal Growth Factor #8916 (100 ng/ml, 30 min) in the presence or absence of calf intestinal phosphatase (CIP) plus lambda-phosphatase, using Phospho-Bad (Ser136) (D25H8) Rabbit mAb (upper) or Bad (D24A9) Rabbit mAb #9239 (lower).
    Phospho-Bad Antibody Sampler Kit: Image 9 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, untreated (left) or lambda phosphatase treated (right), using Phospho-Bad (Ser 112) (40A9) Rabbit mAb.
    Phospho-Bad Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of lysates containing Bad Control Proteins #9293, comprised of nonphosphorylated and phosphorylated Bad peptides, using Phospho-Bad (Ser136) (D25H8) Rabbit mAb.
    Phospho-Bad Antibody Sampler Kit: Image 11 Expand Image
    Immunohistochemical analysis of paraffin embedded COS cells untreated (left) or TPA-treated (right), showing induced cytoplasmic staining using Phospho-Bad (Ser112) (40A9) Rabbit mAb.
    Phospho-Bad Antibody Sampler Kit: Image 12 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Phospho-Bad (Ser 112) (40A9) Rabbit Monoclonal Antibody preincubated with control peptide (left) or Phospho-Bad (Ser 112) Blocking Peptide (IHC Specific) #1026 (right).
    Phospho-Bad Antibody Sampler Kit: Image 13 Expand Image
    Immunohistochemical analysis of paraffin-embedded Non-Hodgkin's lymphoma, using Phospho-Bad (Ser112) (40A9) Rabbit mAb.
    Phospho-Bad Antibody Sampler Kit: Image 14 Expand Image
    Flow cytometric analysis of COS cells, untreated (blue) or TPA/Calyculin A treated (green), using Phospho-Bad (Ser112) (40A9) Rabbit mAb.