Design a Western Blot for ZNRF3

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 · Isoform 2 lacks UniProt residues 1–100 of isoform 1, including its annotated signal-peptide region. An antibody recognizing that region may miss isoform 2. Check the antibody epitope before assigning a smaller band to an isoform; the sequence difference alone does not establish its apparent size.

PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.

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 · ZNRF3 is a single-pass membrane protein, and the supplied observed band is about 130 kDa. Choose transfer conditions that retain and transfer proteins in that size range, then check transfer at about 130 kDa with a total-protein stain.

Blocking · Standard workflow guidance: follow the A02974-1 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 · 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 observed band is about 130 kDa, while the predicted mass is 100.6 kDa. ZNRF3 has a signal peptide at UniProt residues 1–55 and is a single-pass membrane protein, but those features alone do not explain the difference. The supplied features list no glycosylation sites or modified residues, so do not assign the shift to a specific modification.

Use the same sample preparation and membrane-protein recovery approach across samples. ZNRF3 is a cell-membrane protein, so differences in membrane recovery can affect the measured band. Quantify the same validated band and use a loading measure appropriate to the material loaded.

The supplied interaction annotation says RSPO1 promotes ZNRF3 interaction with LGR4 and membrane clearance of ZNRF3. Record RSPO1 exposure and compare matched conditions when interpreting membrane-associated signal. The annotation does not establish a specific Western-blot band change.

First check whether the antibody recognizes UniProt residues 1–100: isoform 2 lacks that region. Compare the band's behavior with the expected membrane-associated signal and validate antibody specificity before assigning it to an isoform. The supplied features do not establish the size or identity of an extra band.