Overview

  • Product name
    Lysosome Fraction Western Blot Cocktail
  • Sample type
    Cell Lysate, Tissue Lysate
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    ab154472 contains 3 Mouse monoclonal antibodies, each targeting a specific organelle marker. The presence of lysosome is detected by anti-LAMP2 antibody; cytosol by anti-GAPDH antibody; and nucleus by anti-Histone H3 (di methyl K9) antibody. This cocktail is suitable for determining the purity of organelle isolates prior to further characterization.

  • Notes

    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 ab154472 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.

Images

  • Sample 1: Marker Sample 2: Human heart homogenate Tissue Lysate, 20 µg Sample 3: HeLa Cell Lysate, 20 µg Sample 4: Mouse heart homogenate Tissue Lysate, 20 µg Sample 5: NIH3T3 Cell Lysate, 20 µg Sample 6: Rat heart homogenate Tissue Lysate, 20 µg Sample 7: H9C2 Cell Lysate, 20 µg Primary: ab154472, 1/250 dilution. Secondary: Anti-mouse IgG VeriBlot for IP secondary antibody (ab131368) – (HRP): 1/1000 dilution Observed bands: LAMP2: 110 kDa GAPDH: 37 kDa Histone H3 (di methyl K9): 17 kDa Notes: When running tissue samples, it is advised to use a secondary antibody that only targets Native IgG such as Veriblot (ab131368). Developed using ECL technique under reducing conditions. Exposure time: 5 min. Run on a 4-20% gradient acrylamide gel, blocking and antibody incubation steps in 5% milk, 20mM 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
    Primary: ab154472, 1/250 dilution
    Secondary: HRP conjugated Goat Anti-Mouse secondary antibody at 1/10000
    Observed Bands: LAMP2: 110 kDa
    GAPDH: 37 kDa
    Histone H3 (di methyl K9): 17 kDa
    Notes: Percentage of antibody signal represents signal present in individual fractions as a proportion of the sum of all three fractions (cytoplasmic, membrane and nuclear). Developed using ECL technique under reducing conditions. HeLa lysate prepared using the Membrane Fractionation Kit (ab139409). Exposure time: 5 mins. Run on a 4-20% gradient acrylamide gel, blocking and antibody incubation steps done in 5% milk, 20mM Tris-HCl, 0.1% TWEEN-20
  • Sample 1: Marker
    Samples 2-5: HeLa (Human epithelial carcinoma cell line) Whole Tissue Lysate – 20 µg
    Lane 1: none
    Lane 2: Anti-LAMP2 antibody – Lysosome Marker
    Lane 3: Anti- GAPDH antibody – Cytosolic Marker
    Lane 4: Anti- Histone H3 (di methyl K9) antibody – Nuclear Marker
    Lane 5: Assembled Lysosome Fraction Antibody Cocktail

    Primary: ab154472, 1/250 dilution
    Secondary:HRP conjugated Goat Anti-Mouse secondary antibody at 1/10000

    Observed bands:
    LAMP2: 110 kDa
    GAPDH: 37 kDa
    Histone H3 (di methyl K9): 17 kDa

    Developed using ECL technique under reducing conditions. Exposure time: 5 mins. on a 4-20% gradient acrylamide gel, blocking and antibody incubation steps done in 5% milk, 20mM Tris-HCl, 0.1% TWEEN-20.

References

ab154472 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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