MitoSciences (MS1097)

Apoptosis and DNA Damage (H2A.X(S139) +cleaved-PARP + Anti-GAPDH) Western Blot Cocktail (ab131385)

Overview

  • Product nameApoptosis and DNA Damage (H2A.X(S139) +cleaved-PARP + Anti-GAPDH) Western Blot Cocktail
  • Species reactivity
    Reacts with: Human

    Does not react with

    Mouse, Rat
  • Product overview

    The DNA Damage and Apoptosis western blot cocktail is designed to study the induction of DNA damage and/or apoptosis in response to various stimuli. The two main components of this cocktail are monoclonal antibodies specific to cleaved-PARP and H2A.X phospho Ser139. H2A.X is a histone H2A family member that is phosphorylated and recruited to sites of double-strand DNA breaks. Poly [ADP-ribose] polymerase 1 (PARP) is a DNA repair enzyme that is cleaved by activated caspases. Combined, these antibodies provide biomarkers of dsDNA breaks (H2A.X phospho Ser139) and apoptosis (cleaved-PARP). An anti-GAPDH antibody is included as a loading control. These three readouts are easily resolved by western blot given their different molecular weights.

  • Notes

    Individual antibodies within the ab131385 cocktail:

    Mouse phospho-H2A.X (pSer139) [9F3] monoclonal, IgG

    Working concentration: 1 µg/ml

     

    Mouse cleaved-PARP [4B5BD2] monoclonal, IgG1

    Working concentration: 1 µg/ml

     

    Mouse GAPDH [3E8AD9] monoclonal, IgG2b:

    Working concentration: 0.1 µg/ml

  • Tested applicationsWBmore details

Properties

  • RelevancePhospho-H2A.X (S139): H2A.X is a variant in the H2A histone family. Upon DNA damage, H2A.X is phosphorylated at Serine 139 and localizes to sites of double-stranded DNA breaks. Also known as gamma-H2A.X, pH2A.X (S139) serves to mark DNA break sites and recruit repair enzymes. In mammals, ATM, ATR and DNA-PK kinases are all known to phosphorylate H2A.X. Cleaved-PARP: PARP-1 is nuclear DNA repair enzyme as component of base excision repair complex. In apoptosis, PARP-1 is cleaved by activated caspase-3 between Asp214 and Gly215, resulting in the formation of an N-terminal 24 kDa fragment containing most of the DNA binding domain and a C-terminal 89 kDa fragment containing the catalytic domain. The proteolysis of PARP-1 through this cleavage renders the enzyme inactive and this further facilitates apoptotic cell death. Thus the presence of 89 kDa PARP-1 fragment is considered to be an important biomarker of apoptosis. GAPDH: loading control
  • Cellular localizationNucleus (cl-PARP and pH2A.X) and cytoplasm (GAPDH)
  • Database links

Applications

Our Abpromise guarantee covers the use of ab131385 in the following tested applications.

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

Application Abreviews Notes
WB 1/250. Predicted molecular weight: 15, 36, 89 kDa. (Entire WB procedure to be done in 4% milk/PBS. Note that pH2A.X is ~16 kDa, hence do not run the SDS-PAGE gel too long. Use anti-Mouse-HRP secondary antibody.)

Apoptosis and DNA Damage (H2A.X(S139) +cleaved-PARP + Anti-GAPDH) Western Blot Cocktail images

  • All lanes : Apoptosis and DNA Damage (H2A.X(S139) +cleaved-PARP + Anti-GAPDH) Western Blot Cocktail (ab131385) at 1/250 dilution (Damage and Apoptosis cocktail (ab131384) )

    Lane 1 : Jurkat cell lysate, untreated
    Lane 2 : Jurkat cell lysate, 1h post UV exposure
    Lane 3 : Jurkat cell lysate, 2h post UV exposure
    Lane 4 : Jurkat cell lysate, 4h post UV exposure

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat anti Mouse HRP at 1/3000 dilution

    Predicted band size : 15, 36, 89 kDa
  • All lanes : Apoptosis and DNA Damage (H2A.X(S139) +cleaved-PARP + Anti-GAPDH) Western Blot Cocktail (ab131385) at 1/250 dilution (DNA Damage and Apoptosis cocktail (ab131384) )

    Lane 1 : HeLa cell lysate, untreated
    Lane 2 : HeLa cell lysate, 20 µM Camptothecin 4h treatment
    Lane 3 : HeLa cell lysate, 1 µM Staurosporin 4h treatment

    Lysates/proteins at 15 µg per lane.

    Secondary
    Goat anti Mouse HRP at 1/3000 dilution

    Predicted band size : 15, 36, 89 kDa
  • All lanes :
    Top panel: H2A.X (pSer139) antibody at 1 µg/mL.
    Bottom panel: total H2A.X antibody at a 1/2000 dilution

    Lane 1 : Jurkat cell lysate, untreated
    Lane 2 : Jurkat cell lysate, 4h post UV exposure
    Lane 3 : Jurkat cell lysate, 4h post UV exposure, treated with phosphatase

    Lysates/proteins at 10 µg per lane.

    Secondary
    Top panel: Goat anti Mouse HRP at a 1/3000 dilution
    Bottom panel: Goat anti Rat HRP at a 1/3000 dilution

    Predicted band size : 15, 36, 89 kDa

References for Apoptosis and DNA Damage (H2A.X(S139) +cleaved-PARP + Anti-GAPDH) Western Blot Cocktail (ab131385)

ab131385 has not yet been referenced specifically in any publications.

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