SMAC/Diablo Antibody (9H10) - BSA Free

Western Blot: Smac Antibody (9H10) [NBP1-97590] - Detection of endogenous and over-expressed DIABLO. When analysis of endogenous DIABLO (50 to 100ug total protein per lane) in healthy cells of human as well as mouse origin is performed, only the processed form of mouse DIABLO (NIH3T3 and FDCP1 mouse cell lines) can be detected. The processed form of mouse DIABLO is detectable when over-expressed in human 293T cells serving as a positive control. The HA-tagged mDIABLO migrates at a slightly higher molecular weight than endogenous wild type mouse DIABLO (mouse testes).

Immunocytochemistry/Immunofluorescence: Smac Antibody (9H10) [NBP1-97590] - Mouse Smac/DIABLO is associated with the mitochondria when overexpressed in human HeLa cells, nuclei are counterstained with propidium iodide (PI).

Immunohistochemistry-Paraffin: Smac Antibody (9H10) [NBP1-97590] - Detection of mouse Smac/DIABLO by clone 9H10.

Product Discontinued

SMAC/Diablo Antibody (9H10) - BSA Free Summary

• Immunogen: Recombinant mouse Smac/DIABLO (aa 55-237).
• Isotype: IgG2a
• Clonality: Monoclonal
• Host: Rat
• Gene: DIABLO
• Purity: Protein G purified

Applications/Dilutions

• Dilutions:
  • ELISA 1:100-1:2000
  • Flow Cytometry 1:10-1:1000
  • Immunocytochemistry/ Immunofluorescence 1:10-1:500
  • Immunohistochemistry 1:10-1:500
  • Immunohistochemistry-Frozen 1:10-1:500
  • Immunohistochemistry-Paraffin 1:10-1:500
  • Immunoprecipitation 1:10-1:500
  • Western Blot 1:100-1:2000
• Application Notes: This antibody is useful in ELISA, Flow Cytometry, Immunocytochemistry, Immunohistochemistry (frozen sections, paraffin sections), Immunoprecipitation, Western Blot, For the detection of human Smac/DIABLO use mAb to Smac/DIABLO (human) (10G7) (Product number NBP1-97604).

Reactivity Notes

Mouse

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.02% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Protein G purified

Notes

Method used for WB: Recommended dilution for MAb to Smac/DIABLO (clone 9H10): 1: 500 to 1:1000 for 4-6 h at room temperature or 4C o/n, followed by incubation with an anti-rat IgG HRP-conjugated antibody at a dilution 1:2000 to 1:5000. Exposure time was 10-30 mins.
Technical note: Only secondary anti-rat IgG antibodies of the highest quality are recommended for use with 9H10 in Western blot (PAb to Rat IgG (Mouse Ig adsorbed)

Method used for ICC: MAb to Smac/DIABLO (clone 9H10) was used at a dilution of 1:100 (in C, phase contrast in A, staining with PI in B, overlay with PI shown in D), but the optimal concentration needs to be determined individually. Cells were fixed with 4% PFA and permeabilized with 0.1-0.3% saponin, followed by incubation with an anti-rat-ALEXA490 conjugated antibody (Molecular Probes). Staining with a negative control (rat IgG) is shown in E to H.

Method used for IHC-P: Freshly cut mouse tissues were fixed, paraffin embedded and cut. Before staining the tissue sections were dewaxed and gradually rehydrated. Tissue sections were treated with 0.3% hydrogen peroxide in PBS to block the endogenous peroxidase activity. The cells were then permeabilized by incubation in 0.2% Triton X-100 and non-specific staining was prevented by incubation with 2.5% horse serum for 1 hr at room temperature. Sections were incubated in a humidified chamber with clone 9H10 at 10 ug/ml for 1 hr at room temperature. After washing in PBS containing 0.2% Triton X-100, tissues were incubated with HRP conjugated goat anti-rat IgG. To enhance the signal, the tissues were incubated with biotinylated goat anti-rat IgG. This was followed by incubating with ABC reagent and finally the sections were stained with DAB according to the manufacturer's instructions. Sections were counterstained with hematoxylin, and dehydrated in graded concentrations of alcohol and histolene before mounting.

Alternate Names for SMAC/Diablo Antibody (9H10) - BSA Free

• 0610041G12Rik
• Diablo
• diablo, IAP-binding mitochondrial protein
• Direct IAP-binding protein with low pI
• mitochondrial Smac protein
• Second mitochondria-derived activator of caspase
• SMAC
• SMACmitochondrial

Background

Smac/DIABLO is a mitochondrial protein which activates various forms of apoptosis. Specifically, Smac exits the mitochondria and enters the cytosol where it initiates apoptosis by neutralizing members of the IAP family of apoptosis inhibitory proteins. Mitochondrial-mediated apoptosis is important in animal development and tissue homeostasis, with alterations resulting in a range of malignant disorders. Upon apoptotic stimuli, the mitochondrial proteins cytochrome c and Smac/DIABLO are released into the cytosol. The release of these proteins, however, occurs via different mechanisms.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for SMAC/Diablo Antibody (NBP1-97590)(4)

Publications using NBP1-97590

• Liu X, Hu J, Li Y et al. Mesenchymal stem cells expressing interleukin-18 inhibit breast cancer in a mouse model. Oncol Lett 2018-05-01 [PMID: 29725393]
• IA Camacho et al. Treatment of mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to aryl hydrocarbon receptor-dependent nuclear translocation of NF-kappaB and expression of Fas ligand in thymic stromal cells and consequent apoptosis in T cells. J. Immunol. 175, 90 (175). [PMID: 15972635]
• A Tikoo et al. Tissue distribution of Diablo/Smac revealed by monoclonal antibodies. Cell Death Differ. 9, 710. 2002-01-01 [PMID: 12058276]
• T Kataoka et al. Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension. J. Biol. Chem. 276, 19548. 2002-01-01 [PMID: 11262395]

Bioinformatics

• Gene Symbol: DIABLO