Anti-ATG8 antibody (ab4753)

Western blot - ATG8 antibody (ab4753)

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 reactivity of anti-APG8 (anti-Apg 8) with other UBLs. This data indicates that anti-ATG8 (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.

Western blot - ATG8 antibody (ab4753)

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.

This image is courtesy of an anonymous abreview.