TY  - JOUR
TI  - Quantification of Folded and Misfolded Proinsulin Forms Using Nonreducing SDS-PAGE and Proinsulin-Specific Immunoblotting
AU  - Arunagiri, Anoop 
AU  - Jang, Insook 
AU  - Itkin-Ansari, Pamela 
AU  - Kaufman, Randal J.
AU  - Arvan, Peter 
VL  - 15
IS  - 11
PY  - 2025
DA  - 2025/06/05
SP  - e5337
C1  - Bio-protocol 2025;15:e5337
DO  - 10.21769/BioProtoc.5337
UR  - https://doi.org/10.21769/BioProtoc.5337
AB  - We have observed that some proinsulin molecules in pancreatic islets and beta cell lines have incomplete or improper intramolecular disulfide bonds. These misfolded monomers can form intermolecular disulfide-linked complexes. Accurately quantifying the fraction of proinsulin molecules contained in these complexes is challenging. By proinsulin immunoblotting after nonreducing SDS-PAGE, the signal for disulfide-linked complexes can exceed the total proinsulin signal detected after reducing SDS-PAGE (i.e., overestimating the abundance of misfolded species due to antibody affinity differences). However, after modification of the SDS-PAGE and electrotransfer protocol, we have been able to more accurately estimate the fraction of proinsulin monomers folded to the native state, as well as misfolded proinsulin monomers and disulfide-linked complexes. Our improved technique offers the ability to obtain a more precise assessment of proinsulin misfolding under different environmental conditions in beta cells and normal islets and in diabetes.
KW  - Proinsulin misfolding
KW  - Quantitation
KW  - Disulfide bonds
KW  - Beta cells
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False