Atg8p (ATG8) antibody

Details for Product No. ABIN116952
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Antigen
Reactivity
Yeast
(1)
Host
Rabbit
(2)
Clonality
Polyclonal
Application
ELISA, Western Blotting (WB)
(2), (2)
Pubmed 5 references available
Catalog no. ABIN116952
Quantity 0.5 mg
Price
390.50 $   Plus shipping costs $45.00
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Immunogen This purified antibody was prepared from rabbit serum after repeated immunizations with recombinant yeast APG8 protein.
Specificity This product is an IgG fraction antibody purified from monospecific antiserum by a multi- step process which includes delipidation, salt fractionation and ion exchange chromatography followed by extensive dialysis against the buffer stated above. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Rabbit Serum.
Purification Multi-step process
Alternative Name APG8 / ATG8
Background Ubiquitin-like proteins fall into two classes: the first class, ubiquitin-like modifiers (UBLs) function as modifiers in a manner analogous to that of ubiquitin. Examples of UBLs are SUMO, Rub1 (also called Nedd8), Apg8 and Apg12. Proteins of the second class include parkin, RAD23 and DSK2, are designated ubiquitin-domain proteins (UDPs). These proteins contain domains that are related to ubiquitin but are otherwise unrelated to each other. In contrast to UBLs, UDPs are not conjugated to other proteins. Apg8 is required for autophagy (intracellular bulk protein degradation) in yeast. Starved yeast cells take up their own cytoplasm into vacuoles through autophagic bodies. Autophagic bodies form a double-membraned structure called the autophagosome, which subsequently fuses with the vacuole/lysosome. This process similar in mammals. Two sets of genes, APG and AUT, have been identified with this process, and are responsible for two ubiquitin-like systems Apg12 and Apg8, respectively. Apg12 is synthesized in its mature form and seems to have one target, Apg5. Almost all Apg12 molecules are conjugated with Apg5. Aut2/Apg4 processes the Apg8/Aut7 system at its carboxy-terminal region. Apg8 exists in two forms, one is membrane bound through a phospholipid. Lipidation/ activation of Apg8 is mediated by Apg7 and transferred to Apg3 and finally forms a conjugate with phosphatidyl- ethanolamine (PE). Apg4 cleaves Apg8-PE, releasing Apg8 from membrane. Morphological studies show that Apg8 localizes on the membrane of intermediate structures of the autophagosome, this transient association seems to be essential for formation of the autophagosome. Both Apg12 and Apg8 are highly conserved, with apparent homologues in the worm, mammals and plants. In higher eukaryotes, Apg8 consists of a multigene family.
Alternate names: AUT7, Autophagy-related protein 8, Autophagy-related ubiquitin-like modifier ATG8, CVT5, Cytoplasm to vacuole targeting protein 5
Gene ID 852200
NCBI Accession NP_009475.1
UniProt P38182
Application Notes This purified polyclonal antibody reacts with yeast APG8 by Western blot and ELISA. Although not tested, this antibody is likely functional in Immunohistochemistry andimmunoprecipitation. This antibody using the specified conditions may recognize otherprominent intrinsic bands (UBLs or their conjugates). Other intrinsic bands are readilydetectable in yeast lysates at lower antibody dilutions. Recommended Dilutions: For Immunoblotting a 1: 4,000 to 1: 8,000 dilution isrecommended. A 14 kDa band corresponding to yeast APG8 is detected. Most yeast cell lysates can beused as a positive control without induction or stimulation. For ELISA a 1: 20,000 to1: 100,000 dilution is recommended. Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user.
Restrictions For Research Use only
Format Lyophilized
Reconstitution Restore with 0.1 mL of deionized water (or equivalent).
Concentration 5.0 mg/mL (by UV absorbance at 280 nm)
Buffer 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2 with 0.01% (w/v) Sodium Azide as preservative
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice Avoid repeated freezing and thawing. Dilute only prior to immediate use
Storage 4 °C/-20 °C
Storage Comment Store vial at 2-8°C prior to restoration. For extended storage add glycerol to 50% and then aliquot contents and freeze at -20°C or below. Centrifuge product if not completely clear after standing at room temperature. This antibody is stable for one month at 2-8°C as an undiluted liquid.
Expiry Date 12 months
Supplier Images
anti-Atg8p (ATG8) antibody Immunoblot of APG8 fusion protein. Anti-APG8 antibody generated by immunization with recombinant yeast APG8 was tested by immunoblot with other anti-UBL antibodies against E.coli lysates expressing the APG8-GFP fusion protein. 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 APG8 using a 1:4,000 and 1:8,000 dilution of ROCKLAND's IgG fraction of Rabbit-anti-APG8 (Yeast) followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG MX (code # 611-103-122). 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 reactivity of anti-APG8 with other UBLs. This data indicates that anti-APG8 is highly specific and does not cross react with other UBLs. A chemiluminescence system was used for signal detection (Roche). Other detection systems will yield similar results. Data contributed by M. Malakhov, www.lifesensors.com, personal communication.
anti-Atg8p (ATG8) antibody (2) Conjugation pathways for ubiquitin and ubiquitin-like modifiers (UBLs). Most modifiers mature by proteolytic processing from inactive precursors (a, amino acid). Arrowheads point to the cleavage sites. Ubiquitin is expressed either as polyubiquitin or as a fusion with ribosomal proteins. Conjugation requires activating (E1) and conjugating (E2) enzymes that form thiolesters (S) with the modifiers. Modification of cullins by RUB involves SCF(SKP1/cullin-1/F-box protein) /CBC(cullin-2/elongin B/elonginC) -like E3 enzymes that are also involved in ubiquitination. In contrast to ubiquitin, the UBLs do not seem to form multi-UBL chains. UCRP(ISG15) resembles two ubiquitin moieties linked head-to-tail. Whether HUB1 functions as a modifier is currently unclear. APG12 and URM1 are distinct from the other modifiers because they are unrelated in sequence to ubiquitin. Data contributed by S.Jentsch, see references below.
Background publications Liakopoulos, Doenges, Matuschewski et al.: "A novel protein modification pathway related to the ubiquitin system." in: The EMBO journal, Vol. 17, Issue 8, pp. 2208-14, 1998 (PubMed).

Kirisako, Baba, Ishihara et al.: "Formation process of autophagosome is traced with Apg8/Aut7p in yeast." in: The Journal of cell biology, Vol. 147, Issue 2, pp. 435-46, 1999 (PubMed).

Jentsch, Pyrowolakis: "Ubiquitin and its kin: how close are the family ties?" in: Trends in cell biology, Vol. 10, Issue 8, pp. 335-42, 2000 (PubMed).

Ohsumi: "Molecular dissection of autophagy: two ubiquitin-like systems." in: Nature reviews. Molecular cell biology, Vol. 2, Issue 3, pp. 211-6, 2001 (PubMed).

Mariño, Uría, Puente et al.: "Human autophagins, a family of cysteine proteinases potentially implicated in cell degradation by autophagy." in: The Journal of biological chemistry, Vol. 278, Issue 6, pp. 3671-8, 2003 (PubMed).

Hosts (2)
Reactivities (1)
Applications (2), (2)
Request Want additional data for this product?

The Independent Validation Initiative strives to provide you with high quality data. Find out more

Order hotline:

  • +1 404 474 4654
  • +1 888 205 9894 (TF)
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