Overview

  • Product name

    Membrane Fraction WB Cocktail
  • Sample type

    Cell culture extracts, Tissue Extracts, Cell Lysate
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    ab140365 contains 5 mAbs each targeting proteins located in different compartments of the cell. The presence of plasma membrane is shown by Anti-Sodium Potassium ATPase; endoplasmic reticulum is detected by GRP78; mitochondrial by ATP5A; cytosol by Anti-GAPDH; and nucleus by Anti-Histone H3 (di methyl K9). This cocktail is suitable for detecting the purity of cellular fractions using kit ab139409.

  • Tested applications

    Suitable for: WBmore details

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 200 µl
    2500X HRP Conjugated Secondary Antibody Cocktail 1 x 60µl
    250X Membrane Fraction Western Blot Cocktail 1 x 200µl
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab140365 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

  • Performed under reducing conditions. All blocking and antibody incubation steps were done in 5% milk in TBST. Developed using the ECL technique. Exposure time: 5 mins.

    Sample 1: Marker
    Samples 2-5: Hela Cell Lysate – 20 µg

    Primary:
    Lane 1: none
    Lane 2: Anti- Sodium Potassium ATPase antibody – Plasma Membrane Marker
    Lane 3: Anti-GRP78 antibody – Endoplasmic Reticulum Marker
    Lane 4: Anti- ATP5a antibody – Mitochondrial Membrane Marker
    Lane 5: Anti- GAPDH antibody – Cytosolic Marker
    Lane 6: Anti- Histone H3 (di methyl K9) antibody – Nuclear Marker
    Lane 7: Assembled Membrane Antibody Cocktail

    Secondary:HRP conjugated secondary antibody cocktail

    Predicted/observed band sizes:
    Sodium Potassium ATPase = 112 kDa/100 kDa
    GRP78 band size: 78 kDa / 75 kDa
    ATP5A band size: 60 kDa / 60 kDa
    GAPDH band size: 37 kDa / 38 kDa
    Histone H3 (di methyl K9) band size: 17 kDa / 17 kDa
  • Developed by IR scanning. HeLa cell lysate prepared using Kit ab139409. Exposure time: 5 mins . Samples run on a 4-20% gradient acrylamide gel. All blocking and antibody incubation steps were done in 5% milk, 20mM Tris-HCl, 0.1% TWEEN-20
    Lane 1: Marker
    Lane 2 : HeLa Whole Cell Lysate
    Lane 3 : HeLa Cytosolic Fraction Lysate
    Lane 4 : HeLa Membrane Fraction Lysate
    Lane 5 : HeLa Nuclear Fraction Lysate
    Primary antibody: Membrane Fraction Western Blot cocktail
    Secondary: IR800 GAM (1/10000) and IR690 GAR (1/10000)
    Predicted/Observed band sizes:
    Sodium Potassium ATPase: 112 kDa/100 kDa
    GRP78: 78 kDa/75 kDa
    ATP5A: 60 kDa/60 kDa
    GAPDH: 37 kDa/37kDa
    Histone H3 (di methyl K9): 17 kDa/17kDa
    Notes: Percentage of antibody signal (Y axis) represents the 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. on a 4-20% gradient acrylamide gel, blocking and antibody incubation steps done in 5% milk, 20mM Tris-HCl, 0.1% TWEEN-20
    Lane 1: Marker
    Lane 2 : HeLa Whole Cell Lysate
    Lane 3 : HeLa Cytosolic Fraction Lysate
    Lane 4 : HeLa Membrane Fraction Lysate
    Lane 5 : HeLa Nuclear Fraction Lysate
    Primary antibody: Membrane Fraction Western Blot cocktail
    Secondary: HRP conjugated secondary antibody cocktail
    Predicted/Observed band sizes:
    Sodium Potassium ATPase: 112 kDa/100 kDa
    GRP78: 78 kDa/75 kDa
    ATP5A: 60 kDa/60 kDa
    GAPDH: 37 kDa/37kDa
    Histone H3 (di methyl K9): 17 kDa/17kDa
    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)
  • Samples run on a 4-20% gradient acrylamide gel. Performed under reducing conditions. All blocking and antibody incubation steps were done in 5% milk, 20mM Tris-HCl, 0.1% TWEEN-20. Developed using the ECL technique. Exposure time: 5 mins.

    Lane 1: Marker
    Lane 2: Human heart homogenate Lysate – 20 µg
    Lane 3: Hela Cell Lysate – 20 µg
    Lane 4: Mouse heart homogenate Lysate - 20 µg
    Lane 5: NIH3T3 Cell Lysate – 20 µg
    Lane 6: Rat heart homogenate Lysate – 20 µg
    Lane 7: H9C2 Cell Lysate – 20 µg

    Primary antibody: Membrane Fraction Western blot cocktail at 1/250 dilution

    Secondary antibody: HRP conjugated secondary antibody cocktail at 1/2500 dilution

    Predicted/observed band sizes:
    Sodium Potassium ATPase = 112 kDa/100 kDa
    GRP78 band size: 78 kDa / 75 kDa
    ATP5A band size: 60 kDa / 60 kDa
    GAPDH band size: 37 kDa / 38 kDa
    Histone H3 (di methyl K9) band size: 17 kDa / 17 kDa

References

This product has been referenced in:

  • Foryst-Ludwig A  et al. Adipose Tissue Lipolysis Promotes Exercise-induced Cardiac Hypertrophy Involving the Lipokine C16:1n7-Palmitoleate. J Biol Chem 290:23603-15 (2015). WB ; Mouse . Read more (PubMed: 26260790) »
  • Bohuslavova R  et al. Partial deficiency of HIF-1a stimulates pathological cardiac changes in streptozotocin-induced diabetic mice. BMC Endocr Disord 14:11 (2014). WB ; Mouse . Read more (PubMed: 24502509) »
See all 2 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Question
Answer

The primary antibodies in the primary cocktail were raised in either rabbit or mouse.

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Answer

Yes, for ab140365 both the primary and secondary antibodies are included in the kit.

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Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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