This is a knockout-validated antibody summary, based on the publications "Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo" [2] (discussed below), "Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9" [1] (see Figure 1 here or figure 1e in the article), "Atg5 Disassociates the V1V0-ATPase to Promote Exosome Production and Tumor Metastasis Independent of Canonical Macroautophagy" [3] (western blot, see figure 1h in the article) and "NIPSNAP1 and NIPSNAP2 Act as "Eat Me" Signals for Mitophagy" for western blot knockout validation (figure 5a) [4]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

Figure 1. Western blot of ATG7 antibody with ATG7 knockout H4 cell lysate and control cell lysate. Adapted from [1].

Rabbit monoclonal IgG

Company: Cell Signaling Technology

Antibody: Atg7

Catalog number: 8558

Summary: Rabbit monoclonal IgG against a synthetic peptide corresponding to residues near the amino terminus of human Atg7 protein. Reacts with human, mouse, and rat. It is predicted to react based on 100% sequence homology with monkey, and bovine. Suitable for western blot and immunoprecipitation.

Western blot

Wild-type, heterozygous and homozygous ATG7-knockout DLD1 cell lines. Cells were lysed in RIPA (Teknova) with sodium dodecyl sulphate (SDS, Sigma) added to 1% final concentration, and protease inhibitor tablets (Roche).

5% non-fat dry milk (Biorad) in PBS with 0.2% Tween-20 (Teknova).

1:1,000 dilution in blocking solution at 4 C overnight.

HRP-conjugated secondary antibodies diluted in blocking solution at room temperature for 1 h.

Pico or West Femto Super Signal ECL reagents (Pierce) and film (GE Healthcare)

Western blot of the indicated proteins in ATG7 wild-type (+/+), heterozygous (+/−) or homozygous knockout (−/−) DLD1 clones 1 and 2. Please see Figure 4d in the article [2].