Design a Western Blot for PER1

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 · Four isoforms are listed. Relative to the canonical 1290-residue sequence, isoform 2 replaces residues 1–32 and 822–875 and lacks residues 876–1290. An antibody targeting the missing region would not detect isoform 2. These are UniProt canonical coordinates; check the antibody epitope before assigning bands to isoforms.

PTM · UniProt lists phosphothreonine 121 and phosphoserines 122 and 126 phosphorylated by CSNK1E, plus phosphoserines 661, 663, 704, 815, 979 and 980. These are canonical UniProt coordinates, which may differ from antibody or paper numbering. Confirm the antibody’s target site; listed phosphorylation alone does not establish a visible shift.

PER1 has listed phosphorylation sites, so assess blocking conditions using the specific phospho-PER1 antibody. Compare target signal and background under otherwise matched conditions, and confirm that the antibody recognizes the intended UniProt site. The supplied features do not specify an optimal blocker.

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 · PER1 has a predicted mass of 136.2 kDa and a supplied observed band near 200 kDa. Choose transfer conditions that retain and transfer proteins in that size range, then check transfer efficiency near 200 kDa. The UniProt features do not establish one preferred transfer method.

Blocking · Standard workflow guidance: follow the A00876 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 · PER1 is part of the circadian core oscillator and occurs in both nucleus and cytoplasm. Compare samples collected at matched time points and process corresponding cell fractions consistently. Quantify the same identified PER1 band across samples; consider separate measurements of nuclear and cytoplasmic signal when studying localization.

Interpretation · The supplied observed band is approximately 200 kDa, whereas the predicted mass is 136.2 kDa. PER1 has multiple phosphorylation sites and isoforms, but those features alone do not establish the cause of this difference. Verify band identity before assigning the higher apparent mass to a specific modification.

Check whether each band matches the antibody epitope and the listed isoforms before assigning identity. Isoform 2 lacks canonical residues 876–1290, including listed phosphoserines 979 and 980. PER1 phosphorylation is documented, but a site’s presence alone does not explain any particular extra band or mobility shift.

Yes. UniProt places PER1 in the nucleus and cytoplasm and describes nucleocytoplasmic shuttling associated with oscillator proteins or phosphorylation. PER1–PER2 heterodimer formation retains PER1 in the cytoplasm. When comparing fractions, measure nuclear and cytoplasmic PER1 separately and keep fraction preparation consistent.