Overview

  • Product name
    Mitochondria Fraction Western Blot Cocktail
  • Sample type
    Cell culture extracts, Adherent cells, Suspension cells, Tissue Extracts, Cell Lysate, Tissue Homogenate
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    Mitochondria Fraction Western Blot Cocktail (ab139416) contains 3 mAbs each targeting a specific organelle marker. The presence of mitochondria is determined by Anti-ATP5A ; cytosol by Anti-GAPDH; and nucleus by Anti-Histone H3 (di methyl K9). This cocktail is suitable for determining the purity of organelle isolates prior to further characterization.


     


    This product is particularly valuable to researchers working in organelle proteomics. Mass spectrometry is frequently used in this field to determine the protein content of targeted organelle isolates. These isolates are obtained using differential centrifugation, density gradient fractionation, biochemical enrichment, or affinity purification. Unfortunately, the various methods of purification available for organelle isolation are imperfect and leave behind contaminants from undesired regions of the cell. These contaminants are inevitable, but being aware of which contaminants are present is crucial for analysis of mass spectrometry results. The high sensitivity and species cross reactivity of the antibodies in this cocktail will quickly and easily reveal impurities caused by imperfect sample preparation.

  • Tested applications
    Suitable for: WBmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab139416 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 Use at an assay dependent concentration.

Suggested working concentration: 1/250 dilution

Images

  • Developed using the ECL technique; Performed under reducing conditions; Exposure time: 5 mins ; All blocking and antibody incubation steps were done in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20

    Sample 1: Marker
    Samples 2-5: MHH (Mouse heart homogenate) Whole Tissue Lysate – 20 µg

    Primary:
    Lane 1: none
    Lane 2: Anti- ATP5A antibody – Mitochondrial Membrane Marker
    Lane 3: Anti- GAPDH antibody – Cytosolic Membrane Marker
    Lane 4: Anti-Histone H3 (di methyl K9) antibody – Nuclear Membrane Marker
    Lane 5: Assembled Mitochondrial Membrane Antibody Cocktail

    Secondary:ab131368 at 1/1000 dilution

    Predicted ATP5a band size: 60 kDa
    Observed ATP5a band size: 55 kDa

    Predicted GAPDH band size: 37 kDa
    Observed GAPDH band size: 37 kDa

    Predicted Histone H3 (di methyl K9) band size: 17 kDa
    Observed Histone H3 (di methyl K9) band size: 17 kDa
  • Sample Preparation: HeLa cell lysate prepared using the Membrane Fractionation Kit (ab139409); Developed using the ECL technique under reducing conditions; Exposure time: 5 mins; Blocking and antibody incubation steps done in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20

    Lane 1: Marker
    Lane 2 : HeLa Whole Cell Lysate - 20 µL
    Lane 3 : HeLa Cytosolic Fraction Lysate - 20 µL
    Lane 4 : HeLa Membrane Fraction Lysate - 20 µL
    Lane 5 : HeLa Nuclear Fraction Lysate - 20 µL

    All Lanes:
    Anti- ATP5A antibody – Mitochondrial Marker – 1/250 dilution
    Anti- GAPDH antibody – Cytosolic Marker – 1/250 dilution
    Anti-Histone H3 (di methyl K9) antibody – Nuclear Marker – 1/250 dilution

    Secondary: Goat polyclonal to Mouse IgG (ab6789) – H&L – Pre-Absorbed (HRP) at 1/10000 dilution

    Observed ATP5a band size: 55 kDa
    Observed GAPDH band size: 37 kDa
    Observed Histone H3 (di methyl K9) band size: 17 kDa
  • Developed using the ECL technique; Performed under reducing conditions; Exposure time: 3 mins; All blocking and antibody incubation steps done in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20

    Sample 1: Marker
    Sample 2: HHH Whole Tissue Lysate - 20 µg
    Sample 3: Hela Whole Cell Lysate – 20 µg
    Sample 4: MHH Whole Tissue Lysate - 20 µg
    Sample 5: NIH3T3 Whole Cell Lysate – 20 µg
    Sample 6: RHH Whole Tissue Lysate – 20 µg
    Sample 7: H9C2 Whole Cell Lysate – 20 µg

    All Lanes:
    Anti- ATP5A antibody – Mitochondrial Marker – 1/250 dilution
    Anti- GAPDH antibody – Cytosolic Marker – 1/250 dilution
    Anti-Histone H3 (di methyl K9) antibody – Nuclear Marker – 1/250 dilution

    Secondary: ab131368 at 1/1000 dilution

    Observed ATP5A band size: 60 kDa
    Observed GAPDH band size: 37 kDa
    Observed Histone H3 (di methyl K9) band size: 15 kDa

References

ab139416 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"

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