GlycOCATCH is designed to specifically bind mucin-type O-glycosylated proteins and peptides. The affinity resin is based on inactive OpeRATOR® enzyme that has been engineered to bind O-glycosylated proteins and peptides with high affinity. O-glycoproteins and peptides are bound to the resin at pH 5-8.
GlycOCATCH is provided in a spin column format to allow easy-to-use enrichment of mucin-type O-glycoproteins. The binding is performed in physiological buffers at pH 5-8 and combined with desialylation using SialEXO for 30 min. Due to the strong interaction between the GlycOCATCH resin and O-glycoproteins/peptides, the elution is performed with 8 M urea. Alternatively, the elution can be performed with the included, active OpeRATOR enzyme, that digests the bound O-glycoproteins N-terminally of the O-glycan sites. In this case, peptides are recovered under native conditions. Since asialylated O-glycans on O-glycoproteins are bound efficiently to the resin, the sialidase mix SialEXO® is included in the box.
Binding Capacity and Unit Definitions
50 μl of the GlycOCATCH resin binds ≥ 50 μg asialylated etanercept when incubated in 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl at room temperature for 30 min.
One unit of SialEXO hydrolyzes sialic acids from ≥ 90% 1 µg glycoprotein (fetuin) when incubated in 20 mM Tris pH 6.8 at 37°C for 2h.
One unit of OpeRATOR digests ≥ 90% of 1 µg glycoprotein (TNFαR) when incubated together with one unit of SialEXO in 20 mM Tris pH 6.8 at 37°C for 2 h.
Content and Storage
The GlycOCATCH box (G3-OC6-002) includes:
4 Microspin columns containing GlycOCATCH resin supplied in 20% EtOH, with no preservatives added. Each column includes sufficient material to bind 50 µg O-glycoprotein
1 vial of SialEXO (200 units) supplied lyophilized in TBS pH 7.6, with no preservatives added.
1 vial of OpeRATOR (200 units) supplied lyophilized in TBS pH 7.6, with no preservatives added.
The GlycOCATCH box is shipped cold. The GlycOCATCH resin should be stored at +4-8 °C upon arrival. Do not freeze the columns! The vials of SialEXO and OpeRATOR should be stored at –20 °C upon arrival. After reconstitution, SialEXO and OpeRATOR are stable for 1 month at +4-8 °C.
