Design a Western Blot for RILP

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. UniProt lists isoforms 1 and 2. Relative to isoform 1, isoform 2 lacks residues 1–210 and replaces WAT at 211–213 with MGA. This substantial sequence difference could affect band position. Check which isoform an antibody recognizes before assigning bands.

Isoform 2 lacks residues 1–210 of isoform 1 and has MGA instead of WAT at 211–213. An antibody whose binding region depends on the missing or changed sequence may recognize the isoforms differently. Check its stated binding region before interpreting a missing or weaker isoform band.

PTM · UniProt lists phosphothreonine at 308 and phosphoserine at 314 and 315, using its sequence coordinates. These sites make phosphorylation a possible consideration, but their presence alone does not establish a detectable band shift. Keep the coordinate convention explicit when comparing an antibody datasheet or paper.

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 A03940 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 · 44.2 kDa is the predicted mass; no observed Western blot band position is supplied. Use it as a reference, but do not assign a band by mass alone. The listed phosphorylation sites do not establish a visible shift or explain any difference between predicted and apparent mass.

RILP is listed as a homodimer, so a higher band could raise that possibility. The feature does not show that the dimer survives Western blot sample preparation. Do not assign a higher band to a dimer from size alone; compare it with the expected monomer region and the blot conditions.

RILP is associated with late endosome, lysosome and phagosome membranes. For comparisons, use the same sample fraction and preparation across samples: a change in recovered RILP could reflect its distribution as well as its abundance. State whether the measurement comes from whole-cell material or a membrane-enriched fraction.

UniProt lists isoform 2 as missing residues 1–210, which provides a specific alternative to check for a smaller band. No signal peptide or propeptide is listed here. Compare the antibody’s binding region with the isoform sequences before assigning the band; size alone cannot establish its identity.