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Biochemistry JoVE (Open Access) Citable · DOI

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

DOI: 10.3791/3749-v
What you'll learn
  • ✓ Apply specific endo- and exoglycosidases to remove glycans from protein samples
  • ✓ Detect glycosylation changes via SDS-PAGE gel mobility shifts
  • ✓ Use Pro-Q Emerald 300 staining to visualize glycosylated proteins
  • ✓ Interpret deglycosylation results from enzymatic treatment experiments
Protocol

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Difficulty
intermediate
Total time
~4–6 hours (enzymatic digestion overnight + gel preparation and staining)
Biosafety
BSL-1

Steps

1
Perform enzymatic deglycosylation of protein samples

Treat glycoprotein samples with specific endo- and exoglycosidases to remove sugar moieties. Incubate under appropriate conditions to allow enzyme digestion.

▶ 01:46
2
Run SDS-PAGE of deglycosylated samples

Load deglycosylated and untreated control samples onto SDS-PAGE gel. Electrophorese to separate proteins and detect shifts in gel mobility caused by glycan removal.

▶ 03:32
3
Stain gel with Pro-Q Emerald 300 reagent

Apply Pro-Q Emerald 300 stain to SDS-PAGE gel to specifically detect and visualize glycosylated proteins. Use appropriate washing and imaging steps for glycan-sensitive detection.

▶ 04:55
4
Analyze deglycosylation results and interpret data

Compare gel banding patterns between enzymatically treated and untreated samples. Document changes in protein mobility and staining intensity to characterize glycan modifications.

▶ 06:49
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