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Lysosome Fraction Western Blot Cocktail (ab154472)

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Lysosome Fraction Western Blot Cocktail Species Cross Reactivity
  • Western Blot analysis of HeLa cell fractions using ab154472
  • Lysosome Fraction Western Blot Cocktail Component Separation.

Key features and details

  • Sample type: Cell Lysate, Tissue Lysate

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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

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 200 µl
    250X Lysosome Fraction Primary Antibody cocktail 1 vial
  • Research areas

    • Kits/ Lysates/ Other
    • Kits
    • Antibody Cocktails
    • Lysosome
  • Function

    Has 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.
  • Pathway

    Carbohydrate degradation; glycolysis; pyruvate from D-glyceraldehyde 3-phosphate: step 1/5.
  • Sequence similarities

    Belongs to the glyceraldehyde-3-phosphate dehydrogenase family.
  • Post-translational
    modifications

    S-nitrosylation of Cys-152 leads to interaction with SIAH1, followed by translocation to the nucleus.
    ISGylated.
    Sulfhydration at Cys-152 increases catalytic activity.
  • Cellular localization

    Cytoplasm > 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.
  • Target information above from: UniProt accession P04406 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • CD107 antigen-like family member B
    • CD107b
    • CD107b antigen
    • G3P_HUMAN
    • GAPDH
    • Glyceraldehyde-3-phosphate dehydrogenase
    • H3/b
    • H3/c
    • H3/d
    • H3/f
    • H3/h
    • H3/i
    • H3/j
    • H3/k
    • H3/l
    • Lysosomal associated membrane protein 2C
    • Lysosome associated membrane glycoprotein 2
    • Lysosome associated membrane glycoprotein 2 precursor
    • MAC3
    • Peptidyl-cysteine S-nitrosylase GAPDH
    see all
  • 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
    • Entrez Gene: 685186 Rat
    • Entrez Gene: 24944 Rat
    • Omim: 309060 Human
    • Omim: 138400 Human
    • SwissProt: P04406 Human
    • SwissProt: P13473 Human
    • SwissProt: P84243 Human
    • SwissProt: Q71DI3 Human
    • SwissProt: P68431 Human
    • SwissProt: P16858 Mouse
    • SwissProt: P17047 Mouse
    • SwissProt: P04797 Rat
    • SwissProt: P17046 Rat
    • Unigene: 544577 Human
    • Unigene: 592355 Human
    • Unigene: 598320 Human
    • Unigene: 304088 Mouse
    • Unigene: 309092 Mouse
    • Unigene: 317779 Mouse
    • Unigene: 343110 Mouse
    • Unigene: 392463 Mouse
    • Unigene: 458138 Mouse
    • Unigene: 458416 Mouse
    • Unigene: 129558 Rat
    • Unigene: 91450 Rat
    see all

Associated products

  • Compatible Secondaries

    • Anti-mouse IgG for IP (HRP) (ab131368)
  • Related Products

    • Organelle Detection Western Blot Cocktail (ab133989)
    • Plasma Membrane Fraction Western Blot Cocktail (ab139413)
    • Endoplasmic Reticulum Fraction Western Blot Cocktail (ab139415)
    • Mitochondria Fraction Western Blot Cocktail (ab139416)
    • Membrane Fraction WB Cocktail (ab140365)

Applications

The Abpromise guarantee

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.
Notes
WB
Use at an assay dependent concentration.

Images

  • Lysosome Fraction Western Blot Cocktail Species Cross Reactivity
    Lysosome Fraction Western Blot Cocktail Species Cross Reactivity
    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
  • Western Blot analysis of HeLa cell fractions using ab154472
    Western Blot analysis of HeLa cell fractions using ab154472
    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
  • Lysosome Fraction Western Blot Cocktail Component Separation.
    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.

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab154472? Please let us know so that we can cite the reference in this datasheet.

ab154472 has not yet been referenced specifically in any publications.

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