Multiple Bands from Isoforms or PTMs

Symptom

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.

1. 1 Multiple protein isoforms or splice variants expressed in cell/tissue model migrate at different molecular weights
2. 2 Glycosylation, SUMOylation, or ubiquitylation add 10-80 kDa mass causing mobility shifts
3. 3 Phosphorylation causes subtle mobility shifts (1-3 kDa) visible as band doublets or triplets
4. 4 Antibody recognizes shared epitope across multiple isoforms or modification states

1. 1 Check Specificity/Sensitivity section on antibody product page for predicted isoform reactivity
2. 2 Reference UniProt database to identify known isoform sequences and predicted molecular weights
3. 3 Use PhosphoSitePlus to identify PTMs (glycosylation, ubiquitylation, phosphorylation) affecting target
4. 4 Treat samples with λ-phosphatase or PNGase F to confirm if bands result from phosphorylation or glycosylation

Related Video (3)

Bio-Rad Laboratories ★ 85

Setting Up and Running Mini-PROTEAN® TGX™ Precast Gels

"Demonstrates SDS-PAGE gel running technique essential for resolving protein isoforms and PTMs that cause multiple bands in IP"

Cell Signaling Technology ★ 80

Western Blotting Protocol

"Western blotting protocol directly related to IP analysis; shows how to detect and interpret multiple protein bands from the same IP experiment"

Cell Signaling Technology ★ 72

How CUT&RUN Profiles Chromatin | Cell Signaling Technology

"CUT&RUN is a chromatin-focused alternative to IP that provides relevant context for understanding immunoprecipitation-based protein detection and interpretation"