Mitochondria Fraction Western Blot Cocktail (ab139416)
Key features and details
- Sample type: Adherent cells, Cell culture extracts, Cell Lysate, Suspension cells, Tissue Extracts, Tissue Homogenate
Overview
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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.
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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at +4°C. Please refer to protocols. -
Components 200 µl 250X Mitochondria Fraction WB Cocktail 1 x 200µl -
Research areas
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Cellular localization
ATP5A: Mitochondrion inner membrane. Cell membrane; Peripheral membrane protein; Extracellular side. GAPDH: Cytoplasm; cytosol. Nucleus. Cytoplasm; perinuclear region. Membrane. Cytoplasm; cytoskeleton Histone 3: Nucleus. Chromosome. -
Alternative names
- ATP synthase alpha chain mitochondrial
- ATP synthase H+ transporting mitochondrial F1 complex alpha subunit 1 cardiac muscle
- ATP synthase H+ transporting mitochondrial F1 complex alpha subunit isoform 2 non-cardiac muscle-like 2
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Database links
- Entrez Gene: 498 Human
- Entrez Gene: 2597 Human
- Entrez Gene: 8290 Human
- Entrez Gene: 11946 Mouse
- Entrez Gene: 14433 Mouse
- Entrez Gene: 65262 Rat
- Entrez Gene: 24383 Rat
- Entrez Gene: 287352 Rat
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Images
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Western Blot for Mitochondrial Antibody Cocktail – Component SeparationDeveloped 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
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Western Blot for Mitochondrial Antibody Cocktail – HeLa FractionsSample 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
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Western Blot for Mitochondrial Membrane Antibody Cocktail – Cross ReactivityDeveloped 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
