Overview

  • Product name

    Endoplasmic Reticulum Fraction Western Blot Cocktail
  • Sample type

    Tissue Extracts, Cell Lysate, Tissue Homogenate, Nuclear Extracts
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    ab139415 contains 3 mAbs each targeting a specific organelle marker. The presence of endoplasmic reticulum is detected by GRP78; 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 ab139415 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.

Primary antibody cocktail = 1/250 dilution

Secondary antibody cocktail = 1/2500 dilution

Images

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

    Samples
    Lane 1: Marker
    Lanes 2-5: Hela Whole Cell Lysate – 20 µg

    Primary antibody:
    Lane 1: None
    Lane 2: Anti-GRP78 antibody – Endoplasmic Reticulum Marker
    Lane 3: Anti- GAPDH antibody – Cytosolic Marker
    Lane 4: Anti- Histone H3 (di methyl K9) antibody – Nuclear Marker
    Lane 5: Assembled Endoplasmic Reticulum Antibody Cocktail

    Secondary:HRP conjugated secondary antibody cocktail at 1/2500

    Predicted GRP78 band size: 78 kDa
    Observed band size: 78 kDa

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

    Predicted Histone H3 (di methyl K9) band size: 17 kDa
    Observed band size: 17 kDa
  • HeLa cell lysates were prepared using the Membrane Fractionation Kit (ab139409), blots were developed using the ECL technique, performed under reducing conditions and exposed for 5 minutes. A 4-15% gradient acrylamide gel was used and blocking and antibody incubation steps were performed in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20.

    Sample lanes:
    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 antibody: ER Fraction WB cocktail at 1/250 dilution
    Secondary antibody: HRP conjugated secondary cocktail at 1/2500.

    Predicted/observed band sizes:
    GRP78: 78 kDa/78 kDa
    GAPDH: 37 kDa/37 kDa
    Histone H3 (di methyl K9): 17 kDa/17 kDa
  • Developed using the ECL technique, performed under reducing conditions and exposed for 3 minutes. All blocking and antibody incubation steps performed in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20.

    Samples:
    1: Marker
    2: Human heart homogenate lysate - 20 µg
    3: HeLa cell lysate - 20 µg
    4: Mouse heart homogenate lysate - 20 µg
    5: NIH3T3 cell lysate - 20 µg
    6: Rat heart homogenate lysate - 20 µg
    7: H9C2 cell lysate - 20 µg

    Primary antibody:ER Fraction WB cocktail at 1/250.
    Secondary antibody: HRP conjugated secondary antibody cocktail at 1/2500.

    Predicted/Observed band sizes:
    GRP78: 78 kDa/75 kDa
    GAPDH: 37 kDa/37 kDa
    Histone H3 (dimethyl K9): 17 kDa/17 kDa

References

ab139415 has not yet been referenced specifically in any publications.

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