Genovis » GlycOCATCH Affinity Purification

Description

GlycOCATCH contains spin columns with inactive OpeRATOR (OgpA) enzyme immobilized on agarose beads. The immobilized inactive enzyme specifically binds core 1 O-glycans for enrichment and purification of mucin-type O-glycosylated proteins and peptides. GlycOCATCH performance is optimal after desialylation using SialEXO and captured O-glycoproteins can be eluted using 8 M urea or active OpeRATOR enzyme. GlycOCATCH is available in spin columns for affinity-capture of 200 ug of O-glycoprotein and supplied with lyophilized SialEXO for preparative desialylation and OpeRATOR enzyme for elution.

Detailed Information

Application	Enrichment of O-glycosylated proteins and peptides
Product Group	GlycOCATCH
Source/Origin	Cloned from A. muciniphila, Produced in E. coli (His-tagged), Engineered OpeRATOR (inactivated), 42 kDa
Product Composition	Immobilized on agarose beads (20% EtOH), OpeRATOR included, SialEXO included
Digestion Site	O-glycan binding
Specificity	Binds desialylated mucin-type O-glycosylated proteins and peptides
Substrates	O-glycoproteins
Reaction Conditions	30 min-2 hour binding at RT according to protocol specifications
Digestion Buffers	Binding Buffer: PBS (pH 7.4), TBS (pH 7.0 – 8.0)
Buffer Compatibility	Optimal binding is obtained in sodium phosphate (10-150 mM) and Tris (50-150 mM) buffers at pH 6.0-8.0., Sodium chloride up to 1 M and/or 0.5 M urea can be added without affecting the performance of the column/purification., Optimal buffer conditions may need to be tested for the glycoprotein solution to be purified.
Additives/Co-factors	Optimal activity on desialylated sample (SialEXO).
Inhibitors	Not tested
Unit Definition	50 µl settled resin binds > 50 µg of desialylated etanercept

FAQs

Can I load a digested protein solution directly onto the GlycOCATCH column?

Yes. IMPORTANT! Before you load your sample make sure that the protease in solution is inactivated and that the pH, buffer, chaotrope and NaCl concentrations are within the recommended ranges according to the instructions.

Does GlycOCATCH bind all O-glycosylated proteins and peptides?

No, GlycOCATCH is designed to bind specifically to mucin type (core 1) O-glycosylated proteins and peptides. The performance is significantly enhanced by removal of sialic acids using SialEXO.

Is it possible to digest the glycoprotein with OpeRATOR first and then enrich the O-glycopeptides on GlycOCATCH?

No, when the glycoprotein is digested with OpeRATOR, the binding capacity of GlycOCATCH is lost.

When and why should I use the OpeRATOR elution procedure?

The OpeRATOR enzyme digests O-glycosylated proteins N-terminally to the serine (S) and threonine (T) glycosylation sites. OpeRATOR therefore facilitates mapping of O-glycosylation sites by LC-MS and as the digested products no longer bind to the GlycOCATCH resin, elution and sample preparation can be achieved in a single step. However, this requires the O-glycosylation sites to be spaced closely together for the OpeRATOR digestion to yield peptides of a size suitable for analysis by LC-MS/MS.