Embedded Files
Experimental Design Schematic
Lectin Glycosylation
Lectin Glycosylation
Being proteins, many lectins are themselves glycosylated
Hypothesis: Lectin glycosylation may cause self-aggregation instead of binding to glycans on the protein of interest
This may hinder accurate glycoprofile predictions
Model Protein: Fetuin B
Model Protein: Fetuin B
Well-studied protein
Protease inhibitor
Clinical applications in diabetes, prostate cancer, pregnancy, etc.
Known structure and glycosylation sites
Lectins needed to define glycoprofile are known (PHA-E, SNA, RCA-1, WGA, DSL)
Image from RCSB Protein Data Bank.
Deglycosylation
Deglycosylation
The first step is to deglycosylate the lectins of interest (LOI)
We used two kits from New England BioLabs, Inc. (NEB)
We ran deglycosylation reaction on all LOI regardless of lectin glycosylation
Glycosylated LOI: SNA, PHA-E, RCA-I
Nonglycosylated LOI: WGA, DSL
PNGase F is a commonly used amidase that removes most N-linked glycans
Contains Chitin-Binding Domain (CBD) tag for removal
Images from QA-Bio
Mix of enzymes (including PNGase F) that remove all N-linked and common O-linked glycans
Direct ELISA
Direct ELISA
Wells are coated with the model protein
Each fluorescein-conjugated lectin is applied to a coated well and unbound lectin is washed away
The fluorescent signal of each well is proportional to the amount of lectin bound to the protein
Page Leader: Jonathan Durnford
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