Design a Western Blot for DEPTOR

Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.

Isoforms · Two isoforms are listed. Isoform 2 lacks residues 42–142 of isoform 1, so a lower band could be consistent with isoform 2. The supplied features do not establish its apparent mass or confirm the identity of any lower band.

PTM · In UniProt coordinates, Met1 is N-acetylated. Phosphorylation is listed at Ser235 (MAPK3), Thr241, Ser244, Ser258, Thr259, Ser263, Ser265, Ser280, Ser282, Ser283, Ser286 and Ser287 (CK1), Tyr289 (SYK), Ser291 (CK1), Ser293 (MTOR), Thr295 (MTOR), Ser297, Ser298, and Ser299 (MTOR). These annotations do not establish a visible mobility change.

DEPTOR associates with mTOR complexes, and MTOR is annotated as phosphorylating Ser293, Thr295, and Ser299. Interpret a phospho-specific signal against total DEPTOR in the same samples. Keep the supplied UniProt coordinates when comparing sites; antibody or paper numbering may use a different convention.

Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.

Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.

Blocking · Standard workflow guidance: follow the A03811-2 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.

Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.

Interpretation · The supplied apparent band is ~48 kDa, close to the 46.3 kDa predicted mass. DEPTOR has many annotated modifications, but their presence alone does not establish a visible shift or explain the difference. Use the observed band as a reference when assessing your blot.

Measure the ~48 kDa total-DEPTOR band consistently across samples and account for the two listed isoforms if your antibody detects both. If measuring phosphorylation, compare the phospho-specific signal with total DEPTOR; a change in phospho signal alone does not establish a change in protein abundance.

DEPTOR is annotated at the lysosomal membrane when associated with mTORC1 and mTORC2. When comparing fractions, check which fraction contains the signal and keep fractionation consistent across samples. The location annotation does not establish that all DEPTOR is membrane-bound.

Compare it with the ~48 kDa observed band and consider whether a lower band fits isoform 2, which lacks isoform 1 residues 42–142. The supplied phosphorylation sites and Met1 acetylation do not, by themselves, identify an extra band or demonstrate a visible shift.