Anti-EEA1 antibody - Early Endosome Marker (ab2900)

Overview

  • Product nameAnti-EEA1 antibody - Early Endosome MarkerSee all EEA1 primary antibodies ...
  • Description
    Rabbit polyclonal to EEA1 - Early Endosome Marker
  • SpecificityDetects a band at 180kDa that represents EEA1 in Western blotting on human cell lines (corresponds to results seen in Mu et al). Also detects a band at 100kDa, we are unsure as to the identity of this band. Immunofluorescence staining of EEA1 in HeLa cells yields a punctate staining pattern consistent with the cytoplasmic distribution of endosomes.
  • Tested applicationsICC/IF, IHC (Methanol fixed), IHC-P, WB, ICC, IP, IHC-FoFr more details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Cow, Dog, Human, Xenopus laevis, Zebrafish, Rhesus monkey, Aplysia
  • Immunogen

    Synthetic peptide derived from within residues 1350 to the C-terminus of Human EEA1.

    (Peptide available as ab14946.)

  • Positive control
    • Rat brain

Properties

Applications

Our Abpromise guarantee covers the use of ab2900 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
ICC/IF Use a concentration of 1 µg/ml.
IHC (Methanol fixed) Use at an assay dependent dilution.
IHC-P Use at an assay dependent dilution.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 180 kDa (predicted molecular weight: 160 kDa).Can be blocked with Human EEA1 peptide (ab14946).
ICC 1/200 - 1/500.
IP Use at an assay dependent dilution.
IHC-FoFr Use at an assay dependent dilution.

Target

  • FunctionBinds phospholipid vesicles containing phosphatidylinositol 3-phosphate and participates in endosomal trafficking.
  • Sequence similaritiesContains 1 C2H2-type zinc finger.
    Contains 1 FYVE-type zinc finger.
  • DomainThe FYVE-type zinc finger domain mediates interactions with phosphatidylinositol 3-phosphate in membranes of early endosomes and penetrates bilayers. The FYVE domain insertion into PtdIns(3)P-enriched membranes is substantially increased in acidic conditions.
  • Cellular localizationCytoplasm. Early endosome membrane.
  • Target information above from: UniProt accession Q15075 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links
  • Alternative names
    • Early endosome antigen 1 antibody
    • Early endosome antigen 1, 162kD antibody
    • Early endosome associated protein antibody
    • EEA 1 antibody
    • EEA1 antibody
    • EEA1_HUMAN antibody
    • Endosome associated protein p162 antibody
    • Endosome-associated protein p162 antibody
    • MST105 antibody
    • MSTP105 antibody
    • ZFYVE2 antibody
    • Zinc finger FYVE domain containing protein 2 antibody
    • Zinc finger FYVE domain-containing protein 2 antibody
    see all

Anti-EEA1 antibody - Early Endosome Marker images

  • Confocal microscopy of fixed primary cultures of rat hippocampal neurons (embryonic day 18) showing immunocytochemical labelling of rabbit polyclonal to EEA1 (ab2900, 1/200; Alexa Fluor 488 1/200; green) and monoclonal mouse anti-b-tubulin-II (1/400; Alexa Fluor 568 1/200; red). Magnification 63x ; insert 180x.
  • ICC/IF image of ab2900 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab2900, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

  • ab2900 Rabbit polyclonal to EEA1 (1/500) was used in fixed HEK cells that had been transfected with Rab5-GFP. Fluorescence microscopy of fixed Rab5-transfected HEK cells showing (left to right) immunocytochemical labelling rabbit polyclonal to EEA1 ab2900 (1/500; Alexa Fluor 568; red), and Rab5-GFP (green) with their overlay (far right). Scale bar = 5µm. Showing little co-localisation as expected. Immunofluorescence microscopy revealed a typically punctuate staining of EEA1 in the HEK cells.

  • Immunolocalisation of EEA1 in HeLa cells using ab2900.
  • All lanes : Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1/1000 dilution

    Lane 1 : HeLa nuclear
    Lane 2 : HeLa whole cell lysate
    Lane 3 : A431 cell lysate
    Lane 4 : Jurkat cell lysate
    Lane 5 : HEK293 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Alexa Fluor anti rabbit at 1/50000 dilution

    Performed under reducing conditions.

    Predicted band size : 160 kDa
    Observed band size : 180 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 100 kDa,41 kDa,50 kDa. We are unsure as to the identity of these extra bands.
  • All lanes : Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1/500 dilution

    Lane 1 : HEK293 Whole Cell lysate
    Lane 2 : HEK293 Whole Cell lysate with Human EEA1 peptide (ab14946) at 1 µg

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size : 160 kDa
    Observed band size : 180 kDa (why is the actual band size different from the predicted?)
  • All lanes : Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1 µg/ml

    Lane 1 : Xenopus laevis lysate
    Lane 2 : Mouse 3T3 cell lysate
    Lane 3 : Mouse brain cell lysate
    Lane 4 : Mouse liver cell lysate
    Lane 5 : Rat brain cell lysate
    Lane 6 : Rat liver cell lysate
    Lane 7 : Dog lysate
    Lane 8 : CHO cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size : 160 kDa
    Observed band size : 100,180 kDa (why is the actual band size different from the predicted?)


    Exposure time : 30 seconds
  • Image courtesy of Human Protein Atlas

        Paraffin embedded sections of human prostate tissue were incubated with ab2900 (1/500 dilution) at room temperature for 30 mins. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

    ab2900 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further images can be found at www.proteinatlas.org

  • ab2900 staining mouse L-cells by ICC/IF.  Cells were formaldehyde fixed, permeabilized in Triton X-100 and incubated with ab2900 diluted 1/1000 for 1 hour at 37°C.  A Cy2® conjugated goat anti-rabbit antibody was used as the secondary.

    See Abreview

References for Anti-EEA1 antibody - Early Endosome Marker (ab2900)

This product has been referenced in:
  • Arany S  et al. Nanoparticle-mediated Gene Silencing Confers Radioprotection to Salivary Glands In Vivo. Mol Ther 21:1182-94 (2013). Mouse . Read more (PubMed: 23511246) »
  • Masuzawa A  et al. Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 304:H966-82 (2013). ICC/IF . Read more (PubMed: 23355340) »

See all 44 Publications for this product

Product Wall

Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Denaturing (10)
Sample Human Cell lysate - whole cell (Hela)
Specification Hela
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Dec 12 2013

Application Immunohistochemistry (PFA perfusion fixed frozen sections)
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Temperature: 20°C
Antigen retrieval step None
Sample Aplysia Tissue sections (buccal ganglia)
Specification buccal ganglia
Permeabilization Yes - triton 1.5%
Fixative Paraformaldehyde
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Ms. Francoise Geffroy

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Submitted Dec 06 2013

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Sample Human Cell (HEK293)
Specification HEK293
Permeabilization Yes - 0.2% triton
Fixative Formaldehyde
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Submitted Dec 04 2013

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (10%)
Sample Mouse Cell lysate - whole cell (fibroblast)
Specification fibroblast
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Nov 21 2013

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (Caco-2 cell)
Specification Caco-2 cell
Fixative Paraformaldehyde
Permeabilization Yes - 0.25% Triton X-100 in TBS
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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Submitted Mar 27 2013

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HepG2)
Specification HepG2
Fixative Paraformaldehyde
Permeabilization Yes - Saponin 0.1%
Blocking step FBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 19°C
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Submitted Feb 06 2013

Application Immunocytochemistry/ Immunofluorescence
Sample Rat Cell (Rat Astrocytes)
Specification Rat Astrocytes
Fixative Paraformaldehyde
Permeabilization Yes - 0.3% TritonX in 0.1% PBS
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 24°C
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Dr. Ruma Raha-Chowdhury

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Submitted Dec 04 2012

Application Western blot
Sample Rhesus monkey Cell lysate - whole cell (Rhesus monkey kidney cells (FRhK-4))
Loading amount 20 µg
Specification Rhesus monkey kidney cells (FRhK-4)
Gel Running Conditions Reduced Denaturing (7 %)
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Nov 19 2012

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (prostate)
Specification prostate
Fixative Paraformaldehyde
Permeabilization Yes - 0.5mg/ml saponin
Blocking step BSA as blocking agent for 20 minute(s) · Concentration: 4% · Temperature: 27°C
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Dr. satyendra tripathi

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Submitted Nov 12 2012

Application Immunocytochemistry/ Immunofluorescence
Sample Rhesus monkey Cell (kidney)
Specification kidney
Fixative Paraformaldehyde
Permeabilization Yes - 0.2% Triton X-100
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 37°C
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Submitted Nov 01 2012

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"