Design a Western Blot for CLTA

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 · Five isoforms are listed: Brain, Non-brain, 3, 4, and 5. Their sequence differences could affect migration, but band position alone cannot identify an isoform. Compare samples and antibody recognition with the relevant isoform sequences.

PTM · UniProt lists phosphoserine at 105, 206, and 236, and N6-acetyllysine at 223 and 242. These are canonical UniProt coordinates. Their presence does not establish a visible shift or explain the observed mass. Residue 105 falls within the segment missing from isoform 5.

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 A05796-3 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 or bands being measured and use the same choice across samples. CLTA has five isoforms with sequence differences, so a single band may not represent every isoform equally. Report the antibody and sample fraction alongside the quantified signal.

Interpretation · The observed band is about 28–30 kDa, compared with a predicted 27.1 kDa. CLTA has splice variants and modified residues, but those features alone do not establish why this band migrates differently. Use the observed range as a reference and verify band identity independently.

Check whether the antibody epitope overlaps residues 73–124, missing in isoform 5; 163–192, missing in Non-brain and 5; or 181–192, missing in isoform 3. Isoform 4 replaces residues 162–180 with S. These are UniProt canonical coordinates; verify the antibody’s numbering convention.

CLTA is listed on the cytoplasmic face of coated pits and vesicles as a peripheral membrane protein, and at the cytoskeleton and spindle. Record the fraction used for each sample and compare like fractions when quantifying its band; distribution can affect the amount recovered.

Check whether the antibody recognizes the listed isoform sequences, then compare the unexpected band with the observed 28–30 kDa range. CLTA’s splice variants and modified residues are possibilities to investigate, but neither establishes the identity of an extra band. Confirm identity independently before assigning it to an isoform or modification.