SDS PAGE band migrates smalle when reduced?! - (Feb/22/2007 )
I am expressing and purifying a protein with theoretical size ~8kDa...it runs ~10kDa under reducing condition on SDS PAGE and ~15kDa non-reduced. Can a protein run smaller when reduced?
Perhaps it is a dimer in its unreduced state on SDS-PAGE?
I used to have problems seperating NGF into monomers for SDS-PAGE. The protein is 14kDa but would run mostly as a dimer unless I alkylated my lysates with Iodoacetamide
If your protein contain SH- groups it is usual event ( intermolecular disulfide bridge formation) for polypeptides like yours. When you use sample buffer with bME they destroyed. Adding iodacetamide in sample buffer without bME prevents intermolecular disulfide bond formation so you can see 8kDa band on SDS -PAGE . When your refold and purify your peptide add 2mM DTT to all buffers to prevent aggregate formation.