Multiple Bands from Isoforms or PTMs (Immunoprecipitation (CST Guide))

Question

Accepted Answer

Multiple specific bands appear at different molecular weights in IP lane. Input control shows same pattern, indicating genuine protein variants rather than non-specific binding. Common causes: 1) Multiple protein isoforms or splice variants expressed in cell/tissue model migrate at different molecular weights 2) Glycosylation, SUMOylation, or ubiquitylation add 10-80 kDa mass causing mobility shifts 3) Phosphorylation causes subtle mobility shifts (1-3 kDa) visible as band doublets or triplets 4) Antibody recognizes shared epitope across multiple isoforms or modification states Solutions: 1) Check Specificity/Sensitivity section on antibody product page for predicted isoform reactivity 2) Reference UniProt database to identify known isoform sequences and predicted molecular weights 3) Use PhosphoSitePlus to identify PTMs (glycosylation, ubiquitylation, phosphorylation) affecting target 4) Treat samples with λ-phosphatase or PNGase F to confirm if bands result from phosphorylation or glycosylation