Wet Lab Work

A wet lab pipeline was developed to deglycosylate lectins and test the change in functionality. The steps for the pipeline were as follows:

1. Enzymatic Deglycosylation of Lectins: 

We utilized PNGase F (NEB Catalog #P0706S), a glycosidase that cuts the innermost GlcNAc and asparagine residue of N-glycans. Hence, PNGaseF would remove virtually all N-glycans from a glycoprotein. For our lectin of interest we used SNA (Vector Laboratories #FL-1301-2) and as a control we used RNAse B (NEB Catalog #P7817S) since it only has a single N-glycan, making for a very simple deglycosylation.

2. SDS-PAGE for Separation of Proteins:

To confirm success and degree of deglycosylation, the glycoprotein samples were run using SDS-PAGE to separate them into bands for analysis.

3. Gel Staining for Proteins and Glycoproteins:

The two gels after SDS-PAGE were stained in different ways: one with Coomassie Blue and one with Glycoprotein Stain.

Coomassie Blue is a protein stain, which basically binds to all proteins regardless of how glycosylated they are. Glycoprotein stain on the other hand only binds to glycoprotein. Hence, a deglycosylated protein would not bind to the stain and show up on the gel.

The comparision between these two gels can now help us observe if deglycosylation was successful or not. The bands present on Coomassie staining that do not show up on the glycoprotein staining would signify successful deglycosylation.

4. ELISA for Functional Analysis of Lectins:

To test whether N-deglycosylation had an effect on the function of SNA, a lectin ELISA was performed using Fetuin B, a glycoprotein that SNA binds to. For this ELISA, SNA was pre-conjugated with fluorescein so SNA bound to Fetuin B would be the source of fluorescence. A decrease in fluorescence signified that deglycosylated proteins lost functionality.