Overview

  • Product name
    Anti-ATG8 antibody
  • Description
    Rabbit polyclonal to ATG8
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, ELISA, Electron Microscopymore details
  • Species reactivity
    Reacts with: Saccharomyces cerevisiae
  • Immunogen

    Recombinant full length protein (S. cerevisiae).

Properties

Applications

Our Abpromise guarantee covers the use of ab4753 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 1/4000 - 1/8000. Detects a band of approximately 14 kDa. This antibody using the specified conditions may recognize other prominent intrinsic bands (UBLs or their conjugates). Other intrinsic bands are readily detectable in yeast lysates at lower antibody dilutions.
IHC-P Use at an assay dependent concentration. PubMed: 20174468
ELISA 1/20000 - 1/100000.
Electron Microscopy Use at an assay dependent concentration. PubMed: 25195618

Target

  • Relevance
    ATG8 is a ubiquitin-like protein in yeast required for autophagy (intracellular bulk protein degradation). Starved yeast cells take up their own cytoplasm into vacuoles through autophagic bodies. ATG8 is requiered for autophagy: modified by the serial action of ATG4, ATG7, and ATG3, and conjugated at the C terminus with phosphatidylethanolamine, to become the form essential for generation of autophagosomes. ATG8 interacts also with the endoplasmic reticulum to Golgi v-SNARE protein BET1 and the vacuolar v-SNARE protein NYV1. ATG8 is highly conserved, with apparent homologues in the worm, mammals and plants. In higher eukaryotes, ATG8 consists of a multigene family.
  • Cellular localization
    Cytoplasmic
  • Database links
    • Alternative names
      • Apg 8 antibody
      • APG8 antibody
      • Atg8p antibody
      • AUT7 antibody
      • CVT5 antibody
      see all

    Images

    • Ab4753, generated by immunization with recombinant yeast ATG8 (or Apg 8), was tested by immunoblot with other anti-UBL (Ubiquitin-like modifier) antibodies against E.coli lysates expressing the ATG8-GFP fusion protein (Apg 8-GFP in both panels)). All UBLs possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL.

      Panel A shows total protein staining using ponceau.

      Panel B shows specific reaction with ATG8 (Apg 8) using a 1:4,000 and 1:8,000 dilution of ab4753 followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG (ab7090). All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4° C. E.coli lysate proteins were separated by SDS-PAGE using a 15% gel.

      Similar experiments (data not shown), where other UBL fusion proteins were separated and probed with this antibody showed no react

    • Anti-ATG8 antibody (ab4753) at 1/1500 dilution + Whole cell lysate prepared from Human huh-7 cells expressing the Saccharomyces cerevisiae protein at 15 µg

      Secondary
      Sheep anti-rabbit IgG conjugated to HRP at 1/7000 dilution

      Observed band size: 15 kDa
      why is the actual band size different from the predicted?


      Exposure time: 4 minutes


      Gel run under denaturing conditions.
      Primary antibody incubated for 10 minutes at 20°C.
      Blocked using 0.5% milk for 1 minute at 20°C.
      Detection method: Western lightning chemiluminescent reagent.

      See Abreview

    • Immuno-TEM using ab4753 at 1/30 dilution to label purified virions of a DNA coccolithovirus. 

      Data from: New Phytol. 2014 Dec;204(4):854-63 (PMID 25195618)

      Daniella Schatz, Adva Shemi, Shilo Rosenwasser, Helena Sabanay, Sharon G Wolf, Shifra Ben-Dor, and Assaf Vardi

    References

    This product has been referenced in:
    • Zhou S  et al. Autophagy contributes to regulate the ROS levels and PCD progress in TMV-infected tomatoes. Plant Sci 269:12-19 (2018). WB . Read more (PubMed: 29606209) »
    • Jo M  et al. Yeast genetic interaction screen of human genes associated with amyotrophic lateral sclerosis: identification of MAP2K5 kinase as a potential drug target. Genome Res 27:1487-1500 (2017). Read more (PubMed: 28596290) »
    See all 6 Publications for this product

    Customer reviews and Q&As

    1-10 of 17 Abreviews or Q&A

    Application
    Western blot
    Loading amount
    35 µg
    Gel Running Conditions
    Non-reduced Non-Denaturing (Native)
    Sample
    Lymnaea stagnalis (Great Pond Snail) Cell lysate - whole cell (CNS (circumesophogeal ganglia))
    Specification
    CNS (circumesophogeal ganglia)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C

    Dan Plas

    Verified customer

    Submitted Dec 10 2013

    Answer

    As far as we are aware, ab4753 has never been tested with samples from P.pastoris. All tested species by our 6 month guarantee are stated on our datasheets, and these are updated as soon as any new information is brought to our attention.

    I am sorry I have not been able to find the the P pastoris ATG8 amino acid sequence on the protein database sites in order to check the alignment with the immunogen. If you have the sequence available, I would be pleased to check this for you.

    If the alignment is high, this will indicate the antibody is likely to detect in P pastoris. If you would like to test the antibody in P pastoris, please do not hesitate to contact me with the sequence prior to the purchase as you may be eligible for our AbTrial testing discount program. <

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    Question
    Answer

    This antibody will primarily detect one band at 14 kDa, but may also recognize other prominent intrinsic bands corresponding to UBLs or their conjugates.

    Read More

    Answer

    Could you send us the protein sequences specific to species in question?We can then check for homology.

    Read More

    Answer

    Thank you very much for contacting us with your question.
    I ran an alignment between the immunogen of ab4753 (full-length S. cerevisiae protein) and the C. reinhardtii autophagy protein sequence, and there is 75% identity between the sequences. It is possible that the antibody will be cross-reactive with Chlamydomonas, but we can't say for sure.
    We don't offer small samples for testing, but we do have a testing discount program if you'd still like to try this antibody. As part of this program, you would first purchase and test ab4753, and then submit an Abreview with your data and protocol. In exchange we will give you a $347 discount (the price of ab4753) on a future purchase.
    I hope that this information will be useful, but if you have further questions or need anything else, please let me know and I'll be happy to help.

    Read More

    Answer

    Muchas gracias por tu respuesta y por enviar las imágenes.

    Efectivamente es posible que el vial concreto que hemos enviado al cliente se haya degradado. Dado que el producto está bajo garantía, estaré encantada de reemplazarle o reembolsarle su importe.

    El único lote actualmente disponible de este anticuerpo es el mismo que recibió el cliente en el último pedido, me temo que no hay más en stock.

    Espero tu respuesta para que me indiques cómo proceder y poder ayudar al cliente.

    Read More

    Answer

    Gracias por contactarnos y mandarnos el cuestionario completo. Lamentablemente no he recibido la imagen adjunta en el cuestionario, por lo que agradecería que si es posible la volvierais a enviar.

    En la datasheet del anticuerpo se indica que éste reconoce otras bandas intensas correspondientes a derivados de ubiquitinas.

    Sin embargo, atendiendo al protocolo seguido, hay una serie de sugerencias que posiblemente mejoraran el rendimiento del anticuerpo, disminuyendo el ruido de fondo:

    - Usar inhibidores enzimáticos, especialmente inhibidores de proteasas, evitan la degradación de la proteína.

    -Para aumentar las condiciones reductoras yo sugeriría aumentar el tiempo de hervido de las muestras a 10 minutos.

    -La dilución usada de anticuerpo es muy baja. Nosotros recomendamos usar diluciones de entre 1/4000- 1/8000. Igualmente, aumentar la dilución del secundario (por ejemplo a 1/6000) puede disminuir el background.

    -Otra sugerencia es cambiar las condiciones de bloqueo. Usando BSA al 5% durante 2 horas.

    Confió en que estas recomendaciones mejoren el resultado. En caso contrario, por favor, no dudéis en contactarme de nuevo.

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    Question
    Answer

    Thank you for contacting us. Unfortunately, I was unable to view the image provided in the word document. Is is possible for you to send a copy of the image separately?

    Based on the customer's protocol, it may be possible to obtain more specific results using less primary antibody. We typically recommend using a 1:4000 dilution. It is also possible that the customer's prolonged washes are removing the blocking agent from the membrane and causing background. For that reason I would recommend performing 3 x 5 minute washes.

    Regarding the lack of signal, it may be due to the lysis in the 100% trichloroacetic acid. Using a gentler lysis buffer such as a 1% NP-40 or 1% Triton X-100 in TBS may help to better preserve the protein epitopes.

    I hope this helps to improve the customer's results. If not, please let me know the original order number and I will be happy to offer a free of charge replacement or credit.

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    Answer

    Thank you for your email.

    I see. Yes, in that case the antibody is indeed not working correctly. I apologize about this.

    I wish you good luck with your research, and please let me know if I can help you with anything else.

    Read More

    Answer

    Thank you for your reply. I'm sorry that the antibody did not work. I was wondering if the ATG8 mutant you were refering to is a deletion mutant (ATG8 protein is absent) or just point or fragmentmutation in the ATG8 protein? In case of the latter, the antibody might still be specific and just detect a band at 27 kDa ( a dimer?).

    In any case, please see below for the refund information.

    Your credit note ID for the refund is xxx.

    I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

    Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

    The credit note ID is for your reference only and does not automatically guarantee the credit.

    I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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    1-10 of 17 Abreviews or Q&A

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

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