PNGase F N-glycan Releasing Kit

The PNGase F N-glycan Releasing Kit (Catalog # EA006) provides PNGase F and necessary buffers for gel analysis as well as mass spectrometry analysis. The receptor binding domain of SARS-CoV-2 spike protein that is fluorescently labeled on N-glycans is provided as a convenient control, allowing for monitoring of the deglycosylation process in gel through fluorescent imaging.

PNGase F N-glycan Releasing Kit

Features

• Gel and mass spectrometry analysis of N‑glycans
• Cy5-RBD fluorescent control to monitor the deglycosylation process through fluorescent imaging via gel electrophoresis
• Protocols are provided to guide further assay optimization
• Removes N-linked glycans from Glycoproteins
• Guaranteed Enzymatic Activity: Enzymatic activity tested in a deglycosylation assay

Kit Contents

• PNGase F Protein
• Cy5-Neu5Ac Labeled RBD Protein (Cy5-RBD)
• PNGase F Assay Buffer
• Denaturing Buffer
• Renaturing Buffer
• 6X SDS Gel Loading Dye

Other Reagents Required

• 37°C incubator
• Microcentrifuge tubes or equivalent
• Pipettes and pipette tips
• Deionized or distilled water
• Equipment to run SDS-PAGE gel
• Fluorescent imager
• Mass Spectrometry Instrumentation

Preparation and Storage

Stability & Storage
Store the unopened product at ≤ -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Background: PNGase F

N-Glycosylation found on the asparagine residue of the sequon NXT/S is one of the most commonposttranslational modifications of proteins in eukaryotic cells. N‑glycans are initiated as high-mannose glycan, which are converted to hybrid and complex N‑glycans during the maturation process in the Golgi apparatus. N‑glycosylation involves in myriad of biological functions such as receptor binding, cell signaling, immune recognition, inflammation, and pathogenicity.

To study these N-glycans, it is essential to release them from proteins. PNGase F is a peptide N‑glycosidase F from Flavobacterium meningosepticum and the most used enzyme to extensively release various N‑glycans, including high-mannose, hybrid and complex types of N‑Glycans, except those N‑glycans with the innermost GlcNAc modified with a core‑3 linked fucose. PNGase F digestion results in the release of entire N‑glycans and conversion of the underlying asparagine residue (N) to an aspartic residue (D), allowing identification of the glycosylation site by mass spectrometry.