Anti-EEA1 antibody - Early Endosome Marker (ab2900)

Overview

  • Product nameAnti-EEA1 antibody - Early Endosome Marker
    See 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 applicationsSuitable for: ICC/IF, IHC (Methanol fixed), IHC-P, WB, ICC, IP, IHC-FoFrmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, 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 Abreviews Notes
ICC/IF Use a concentration of 1 µg/ml.
IHC (Methanol fixed) Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
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).

Abcam recommends using milk as the blocking agent.

ICC 1/200 - 1/500.
IP Use at an assay dependent concentration.
IHC-FoFr Use at an assay dependent concentration.

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.
  • 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

  • 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).

  • All lanes : Anti-EEA1 antibody - Early Endosome Marker (ab2900) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human) Whole Cell Lysate
    Lane 3 : NIH 3T3 (Mouse) Whole Cell Lysate
    Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 5 : HEK293 (Human) Whole Cell Lysate
    Lane 6 : NIH 3T3 (Mouse) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG H&L (HRP) at 1/50000 dilution
    Developed using the ECL technique

    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. We are unsure as to the identity of these extra bands.

    Exposure time : 30 seconds

    This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin (Lanes 1-3) or 3% Milk (Lanes 4-6) before being incubated with ab2900 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

    Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

  • 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.
    Fluorescence detection of secondary antibody.
  • 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

  • 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-β.

  • 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.

  • 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

  • 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?)
    Lane 1 - 2 : EEA1 antibody - Early Endosome Marker (ab2900) at 1/500 dilution, HEK293 Whole Cell lysate at 20 ug Lane 1 : as above Lane 2 : EEA1 peptide (ab14946) at 1 ug Secondary Goat polyclonal to Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution Performed under reducing conditions. Predicted band size : 160kD Observed band size : 180kD (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

    The Western blot shows that ab14944 reacts strongly with mouse 3T3 and CHO cell lysates. Weak cross-reactivity is seen with Xenopus, mouse brain, mouse liver, rat brain and dog lysates. The antibody does not appear to cross-react with rat liver lysate.

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

This product has been referenced in:
  • Bourseau-Guilmain E  et al. Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells. Nat Commun 7:11371 (2016). WB, IHC-Fr, ICC/IF . Read more (PubMed: 27094744) »
  • Raha AA  et al. Neuroprotective Effect of TREM-2 in Aging and Alzheimer's Disease Model. J Alzheimers Dis N/A:N/A (2016). IHC ; Mouse . Read more (PubMed: 27662313) »

See all 65 Publications for this product

Product Wall

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
Username

Ms. Francoise Geffroy

Verified customer

Submitted Dec 06 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
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
Username

Abcam user community

Verified customer

Submitted Mar 27 2013

Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (Acinar epithelial explants in collagen)
Permeabilization Yes - TritonX100 0.025% 10 min RT
Specification Acinar epithelial explants in collagen
Blocking step BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 19°C
Fixative Paraformaldehyde
Username

Dr. Clara Lubeseder-Martellato

Verified customer

Submitted Jun 16 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Tissue lysate - whole (Pancreas or PDAC cells)
Gel Running Conditions Reduced Denaturing (10%)
Loading amount 30 µg
Specification Pancreas or PDAC cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 19°C
Username

Abcam user community

Verified customer

Submitted Jun 14 2016

Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (neuron)
Permeabilization Yes - 0.2%Triton
Specification neuron
Blocking step Serum as blocking agent for 40 minute(s) · Concentration: 5% · Temperature: 22°C
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Nov 30 2015

Application Western blot
Sample Mouse Tissue lysate - whole (BRAIN)
Gel Running Conditions Reduced Denaturing (8%)
Loading amount 50 µg
Treatment alpha synuclein
Specification BRAIN
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Nov 09 2015

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Sample Chicken Cell (Primary oculomotor culture)
Specification Primary oculomotor culture
Permeabilization Yes - PBS + 0.1% Tween20
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Oct 27 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Sample Human Cell (ovarian carcinoma TOV112-D cell line)
Specification ovarian carcinoma TOV112-D cell line
Permeabilization Yes - 0.1% triton X for 5 min at RT
Fixative Paraformaldehyde
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Abcam user community

Verified customer

Submitted Aug 27 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 10 minute(s) · Concentration: 5% · Temperature: 22°C
Sample Rat Cell (hippocampal neuron)
Specification hippocampal neuron
Permeabilization Yes - 2% TritonX100
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Aug 13 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Goat serum 5% + BSA 1% as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Sample Human Cell (U87 Glioblastoma)
Specification U87 Glioblastoma
Permeabilization Yes - triton 0.2%
Fixative Paraformaldehyde
Username

Ms. Francoise Geffroy

Verified customer

Submitted Aug 01 2014

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