Design a Western Blot for ADAMTS9

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 · Yes. Four isoforms are listed. Relative to the canonical UniProt sequence, isoform 4 lacks residues 324..351, isoform 3 changes 1064..1072 and lacks 1073..1935, and isoform 2 changes 1624..1629 and lacks 1630..1935. Check whether the antibody recognizes a region retained in the isoform of interest; these sequence differences do not establish observed band positions.

PTM · UniProt lists N-linked sites at canonical positions 112, 135, 271, 749, 840, 1213, 1267, 1788, and 1806. Positions 112, 135, and 271 lie within the annotated propeptide (19..287). Consider the antibody's recognized region and possible processing when interpreting bands. Site annotations alone do not show that glycosylation causes a visible shift.

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 A04639-1 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 · Define the band and sample fraction before comparing signals. ADAMTS9 is annotated in the endoplasmic reticulum and extracellular compartments, and its isoforms and processing can affect which forms an antibody detects. Quantify a consistently identified band with the same antibody and sample preparation across conditions; do not automatically combine distinct bands as one form.

Interpretation · The observed ~216 kDa band is close to the 216.5 kDa predicted mass. ADAMTS9 also has a signal peptide, propeptide, and nine listed N-linked glycosylation sites. Those features alone do not establish a visible shift or explain the apparent mass. Confirm band identity with an antibody whose recognized region is known.

The canonical UniProt sequence has a signal peptide at 1..18 and a propeptide at 19..287; ADAMTS9 is annotated as a zymogen. A smaller band could be consistent with processing, but these annotations alone cannot identify a band. Check whether the antibody recognizes the propeptide or a region retained after processing.

ADAMTS9 is annotated in the extracellular space, extracellular matrix, and endoplasmic reticulum. Compare suitable cellular and extracellular samples if detection is weak in one fraction. A difference between fractions should be interpreted alongside antibody recognition and the protein's processing annotations.

Consider the annotated signal peptide and propeptide, four isoforms, and N-linked glycosylation sites. In canonical UniProt coordinates, isoforms 2 and 3 lack C-terminal regions 1630..1935 and 1073..1935, respectively. Check whether the antibody recognizes a region present in each candidate form. These features suggest possibilities but do not identify an unexpected band by size alone.