Glycoprotein sizing on the Agilent 2100 bioanalyzer
Glycoproteins often migrate at different sizes than anticipated when analyzed using traditional SDS-PAGE or the Agilent 2100 bioanalyzer due to glycan attachment altering shape and charge-to-mass ratios. This application note details a procedure to address this issue.
The document outlines an experimental protocol where proteins are treated with N-Glycosidase F from a Roche Diagnostics kit to remove oligosaccharides. Following deglycosylation, the samples are analyzed using the Protein 200 Plus LabChip kit on the Agilent 2100 bioanalyzer. Results demonstrate that removing glycans allows proteins to migrate at their expected molecular weights, providing accurate sizing compared to untreated glycoproteins.
Enzymatic deglycosylation removes glycan attachments to allow the protein to migrate at its expected size, enabling accurate molecular weight measurement.
The enzymatic deglycosylation reaction should be performed for 1 hour at 37 °C.
Samples are run on the Novex 12 % Tris—Glycine Pre-Cast gel at 120 V.