Overview

  • Product name

  • Description

    Rabbit polyclonal to EHD1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, IP, ICC/IFmore details
  • Species reactivity

    Reacts with: Rat, Human
    Predicted to work with: Mouse, Rabbit, Cow, Non human primates
  • Immunogen

    Synthetic peptide corresponding to Human EHD1 aa 500 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab87397)

  • Positive control

    • This antibody gave a positive signal in Rat Colon Tissue Lysate and in TE 671 Whole Cell Lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab75886 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 a concentration of 1 µg/ml. Detects a band of approximately 61 kDa (predicted molecular weight: 61 kDa).
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IP Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.

Target

Images

  • IHC image of EHD1 staining in normal human colon formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab75886, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • All lanes : Anti-EHD1 antibody (ab75886) at 1 µg/ml

    Lane 1 : Colon (Rat) Tissue Lysate
    Lane 2 : TE 671 (Human Rhabdomyosarcoma) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 61 kDa
    Observed band size: 61 kDa
    Additional bands at: 63 kDa (possible post-translational modification)



    EHD1 contains potential phosphorylation site (SwissProt) which may explain the higher migrating band.
  • EHD1 was immunoprecipitated using 0.5mg Rat Colon whole tissue extract, 5µg of Rabbit polyclonal to EHD1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Rat Colon whole tissue extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab75886.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 61kDa: EHD1.
  • ICC/IF image of ab75886 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab75886, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:

  • Yu H  et al. The expression of Eps15 homology domain 1 is negatively correlated with disease-free survival and overall survival of osteosarcoma patients. J Orthop Surg Res 14:103 (2019). Read more (PubMed: 30975166) »
  • Gao J  et al. EHD1 confers resistance to cisplatin in non-small cell lung cancer by regulating intracellular cisplatin concentrations. BMC Cancer 16:470 (2016). IHC-P ; Human . Read more (PubMed: 27411790) »
See all 4 Publications for this product

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