Design a Western Blot for PCCA

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 · UniProt lists three isoforms. Relative to the canonical sequence, isoform 2 lacks residues 36–61 and isoform 3 lacks residues 634–680. These deletions could change molecular mass, but the features do not establish where either isoform runs on a gel.

PTM · UniProt lists phosphoserine 252 and multiple acetylated or succinylated lysines, with alternate modifications at lysines 65, 150, 200, and 328. These are canonical UniProt coordinates; antibody or paper numbering may differ. Their presence does not establish a visible band 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 A03395-1 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 · Use the same consistently identified PCCA band across samples and report which band was measured. The three isoforms and listed modifications make band identity relevant, but the supplied features do not show that any specific band represents total PCCA or a particular modification.

Interpretation · The supplied observed band is about 70 kDa, while the predicted mass is 80.1 kDa. PCCA has alternative isoforms and a transit peptide keyword, but these features alone do not establish the cause of this difference. Confirm band identity before assigning a mechanism.

UniProt assigns N6-biotinyllysine at lysine 694, modified by HLCS. Position 694 uses canonical UniProt numbering. Check the numbering convention when comparing an antibody or publication; the listed modification alone does not predict a separate band.

PCCA is located in the mitochondrial matrix and has a transit peptide keyword, but the supplied features give no cleavage coordinates or mature-chain mass. Mitochondrial targeting alone cannot establish why an approximately 70 kDa band differs from the 80.1 kDa prediction.

Check candidate bands against the canonical 80.1 kDa prediction and the isoform deletions at residues 36–61 and 634–680. PCCA also forms a holoenzyme with PCCB, but these features do not identify any particular extra band. Do not assign a band to an isoform, modification, or complex from migration alone.