Lysosome Fraction Western Blot Cocktail (ab154472)
- Datasheet
- References
- Protocols
Overview
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Product nameLysosome Fraction Western Blot Cocktail
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Sample typeCell Lysate, Tissue Lysate
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Species reactivityReacts with: Mouse, Rat, Human
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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.
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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.
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Tested applicationsSuitable for: WBmore details
Properties
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Storage instructionsStore at +4°C. Please refer to protocols.
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Components 200 µl ab154472 - 250X Lysosome Fraction Primary Antibody cocktail 1 vial -
Research areas
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FunctionHas both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively. Participates in nuclear events including transcription, RNA transport, DNA replication and apoptosis. Nuclear functions are probably due to the nitrosylase activity that mediates cysteine S-nitrosylation of nuclear target proteins such as SIRT1, HDAC2 and PRKDC. Modulates the organization and assembly of the cytoskeleton. Facilitates the CHP1-dependent microtubule and membrane associations through its ability to stimulate the binding of CHP1 to microtubules (By similarity). Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in glycolysis that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate.
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PathwayCarbohydrate degradation; glycolysis; pyruvate from D-glyceraldehyde 3-phosphate: step 1/5.
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Sequence similaritiesBelongs to the glyceraldehyde-3-phosphate dehydrogenase family.
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Post-translational
modificationsS-nitrosylation of Cys-152 leads to interaction with SIAH1, followed by translocation to the nucleus.
ISGylated.
Sulfhydration at Cys-152 increases catalytic activity. -
Cellular localizationCytoplasm > cytosol. Nucleus. Cytoplasm > perinuclear region. Membrane. Cytoplasm > cytoskeleton. Translocates to the nucleus following S-nitrosylation and interaction with SIAH1, which contains a nuclear localization signal (By similarity). Postnuclear and Perinuclear regions.
- Information by UniProt
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Alternative names
- CD107 antigen-like family member B
- CD107b
- CD107b antigen
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Database links
- Entrez Gene: 2597 Human
- Entrez Gene: 8290 Human
- Entrez Gene: 8350 Human
- Entrez Gene: 126961 Human
- Entrez Gene: 100042025 Mouse
- Entrez Gene: 14433 Mouse
- Entrez Gene: 16784 Mouse
- Entrez Gene: 24383 Rat
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Associated products
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Compatible Secondaries
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Related Products
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
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Lysosome Fraction Western Blot Cocktail Species Cross ReactivitySample 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 -
Western Blot analysis of HeLa cell fractions using ab154472Lane 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 -
Lysosome Fraction Western Blot Cocktail Component Separation.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.
