SDS-PAGE and MALDI-TOF analysis of PEGylated and unPEGylated SK constructs

All PEGylated and unPEGylated derivatives were checked for their purity and conversion (unPEGylated to PEGylated form) on the SDS-PAGE gel. For this, 5ug of the protein (PEGylated or unPEGylated) was mixed in 5X loading dye (reducing and non-reducing dye separately) and separated on 7.5% polyacrylamide gel. Gel was run slowly in 1X Tris-glycine buffer at 25 mA for 2 hours for fine separation. When gel was over, it was placed with coomassie staining solution (Bio-Rad) for 1h followed by repeatedly washing in destaining solution to clearly visualize the sharp bands. Reference protein ladder (GE-Healthcare) was used to determine the exact protein size. Although, the accurate determination of the molecular weights were determined by MALDI-TOF. For MALDI-TOF analysis, 1 mg/ml protein was desalted in water on 1 ml column containing G-25 Sephadex media. The desalted proteins were analysed on an AB SCIEX instrument (model-AB SCIEX TOF/TOF^TM 5800) for their accurate molecular weights.