Overview

  • Product name
  • Description
    Rabbit polyclonal to EEA1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide:

    GSQGSDLDSSATPINTVDVNNESSSEG

    conjugated to KLH by a C-terminal Cysteine residue linker, corresponding to amino acids 24 - 40 of Human EEA1

  • Positive control
    • whole extract of mouse NIH-3T3 cells; human HeLa and rat NRK cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab50313 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 0.4 - 0.8 µg/ml. Detects a band of approximately 160 kDa.
ICC Use at an assay dependent concentration.
ICC/IF Use a concentration of 5 - 10 µg/ml.

Target

  • Function
    Binds phospholipid vesicles containing phosphatidylinositol 3-phosphate and participates in endosomal trafficking.
  • Sequence similarities
    Contains 1 C2H2-type zinc finger.
    Contains 1 FYVE-type zinc finger.
  • Domain
    The 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 localization
    Cytoplasm. 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

Images

  • HeLa cells fixed and permeabilized with methanol followed by acetone. Fixed cells were stained with 1 µg/mL ab50313."
  • ab50313 staining EEA1 in mouse primary astrocytes by Immunocytochemistry. Cells were fixed with paraformaldehyde, permeabilized with 0.05% saponin and blocking with 10% serum was performed for 20 minutes at 250C. Samples were incubated with primary antibody (1/150: in 10% goat serum / PBS) for 1 hour at 25°C. An Abcam`s ab6939, Cy3®-conjugated goat polyclonal to rabbit IgG (H&L) was used as secondary antibody at 1/200 dilution.

    See Abreview

References

This product has been referenced in:
  • Leiva-Rodríguez T  et al. ATG5 overexpression is neuroprotective and attenuates cytoskeletal and vesicle-trafficking alterations in axotomized motoneurons. Cell Death Dis 9:626 (2018). Read more (PubMed: 29799519) »
  • Acharya M  et al. av Integrins combine with LC3 and atg5 to regulate Toll-like receptor signalling in B cells. Nat Commun 7:10917 (2016). Read more (PubMed: 26965188) »
See all 14 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Question

Thanks for your email. Please find my answers inserted below.

I will be happy to send the replacement ab95195, but I will need the original order number or PO number that ab2074 was received on. Would you be able to provide this information? I've found an order from 2007 going to ***; could this be your order? If so, did the antibody work when it was first received? At what temperature has it been stored?

Yes, it is correct that this is still about our order in 2007. As we discussed last time on the phone, the antibody did not work from the very beginning. Originally, I could not be sure that the antibody was the problem because I did not have transfected cells or control antibody available for a rigorous comparison. We know now that we should have been able to detect endogenous CXCR4 in Hela cells under the conditions used, and we did not in 2007. Therefore, you graciously offered to still send a replacement after this long time. We aliquot our antibodies and store them in two ways: We keep a small aliquot at 4C for immediate testing, another aliquot receives 50% glycerol/PBS and is stored at -20C; the latter seems the best for most antibodies and we prefer it over freezing of multiple small aliquots. Neither aliquot worked. However, I did notice that there was some precipitate at the bottom of the vial when we received it.So, I cannot rule out that is was just the specific batch of CXCR4 antibody you sent that was flawed.

Thank you also for your feedback regarding the conditions of ab24170. Would you be able to submit an Abreview showing your results under these conditions, to help highlight the optimal conditions? You will receive Abpoints in exchange for submitting the review, and these points can be redeemed for a discount on a future purchase. I've also added your feedback to our notes on this antibody.

Will do as soon as I find the time.

I am very sorry to hear that the EEA1 antibody ab50313 did not give satisfactory results, and I can send a replacement of your choice. We have a rabbit monoclonal antibody to this target, ab109110, which is suitable for use with mouse and human samples in ICC/IF:

https://www.abcam.com/EEA1-antibody-EPR4245-ab109110.html

Would this antibody work for you? Are there any other targets that you're interested in besides EEA1?

The data sheet states that ab109110 works for ICC, but there is no image. I would like to hold off with a replacement until I see an image that shows the expected pattern in cultured cells. Also, we spent an enormous amount of time on testing antibodies and had to move on with the project. Thus, we may be more interested in a different epitome in due course.

Thank you again for your response and I look forward to hearing from you. Please let me know if you have any questions or need anything else at this time.

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Answer

Thank you very much for your responses.
I'm sending a free of charge vial of ab95195 on the order ***, which should arrive tomorrow. Please keep me updated about the results using this antibody.
For the replacement of ab50313, I certainly understand that you have spent a lot of time on this target. So, please let me know once you have chosen a replacement and I'll be happy to send that out to you promptly.
Please let me know if you have any questions or if there is anything else that we can do for you.
Have a nice day!

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Question

I promised to get back in touch with you several months ago, my apologies that it has taken me this long. Here is some more informationabout three of your antibodies and my request for a second replacement.

1. I have previously been in contact regarding ab2074, which did not yield specific staining for us under the following conditionsat aconcentration of up to 100 ug/ml (1:200 dilution),while a different antibody (UMB2) gave bright staining:

- cells transfected with human or rat CXCR4 cDNA, PFA-fixed and permeabilized with TX100
- Hela cellsPFA-fixed and permeabilized with TX100 or methanol fixed

You offered a replacement, but I did not yet specify the antibody I would like to receive. I now would like to request anti-APPL antibodyab95195 as a replacement.



2. Information for anti-lamp-1 antibody ab24170: This antibody works well for us in cells of human or rat origin at 5 ug/ml when using PFA fixation followed by saponin permeabilization. This antibody does NOT work in our hands using TX100 or methanol permeabilization. This finding seems to be typical for antibodies directed to the short cytoplasmic tails of lysosomal membrane proteins (we saw it before with two different antibodies of this kind). Note that another customer used Tween and achieved a labeling pattern consistent with that of lysosomes. It would be helpful to include a note in the data sheet that permeabilizations other than with TX100 or methanol (such as saponin) should be tried if standard procedures are not successful (most frequently TX100 or methanol are used) to avoid frustrations or wrong results with this fine antibody.



3. Information for anti-EEA1 antibody ab 50313 and request for replacement (to be specified). We received this antibody as a replacement for ab2900 on 8/30/11. This antibody was also not satisfactory for us, although it gave more specific staining (together with strong nonspecific staining). We found that the reactivity of EEA1 antibodies is much better after short fixations (5 min, 4% PFA) than after longer fixations (>30 min and/or higher temperatures) indicating that some epitopes are sensitive to (over)fixation. While we can obtain some EEA1-specific labelingwith ab50313, as evidenced by coincidence staining with a monoclonal ab to EEA1, there are also many structures in our cells that are similarly bright labeled withab50313 that are negative for mAb staining. Therefore, this antibody is also of no use to as we cannot use it with confidence to identify early endosomes. We have been using Flp-In™-293 cells, a subclone of the commonly used human 293 cells.


Also, please note my new email address.


Best regards,

Read More
Answer

Thank you very much for your reply and for sending all of this information.
I will be happy to send the replacement ab95195, but I will need the original order number or PO number that ab2074 was received on. Would you be able to provide this information? I've found an order from 2007; could this be your order? If so, did the antibody work when it was first received? At what temperature has it been stored?
Thank you also for your feedback regarding the conditions of ab24170. Would you be able to submit an Abreview showing your results under these conditions, to help highlight the optimal conditions? You will receive Abpoints in exchange for submitting the review, and these points can be redeemed for a discount on a future purchase. I've also added your feedback to our notes on this antibody.
I am very sorry to hear that the EEA1 antibody ab50313 did not give satisfactory results, and I can send a replacement of your choice. We have a rabbit monoclonal antibody to this target, ab109110, which is suitable for use with mouse and human samples in ICC/IF:
https://www.abcam.com/EEA1-antibody-EPR4245-ab109110.html
Would this antibody work for you? Are there any other targets that you're interested in besides EEA1?
Thank you again for your response and I look forward to hearing from you. Please let me know if you have any questions or need anything else at this time.

Read More

Answer

Thanks for the update. We certainly appreciate your lab's troubleshooting efforts. Please feel free to reply after your trip.

Read More

Question
Answer

We have previously been in contact regarding ab2074, which was not providing satisfactory results. We had offered a free of charge replacement with a different antibody, but we were waiting to hear from you with your selection. If you have determined which antibody you would like as a replacement, please let us know and we will send it promptly. I wish you the best of luck with your research and please do not hesitate to contact us if you have any further questions.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Mouse Cultured Cells (primary astrocyte)
Specification
primary astrocyte
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.05% saponin
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 08 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Lung carcinoma cell line)
Specification
Lung carcinoma cell line
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton X-100
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Aug 17 2009

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

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