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GlycOCATCH workflow

GlycOCATCH®

GlycOCATCH is an enrichment resin for affinity purification of mucin-type O-glycosylated proteins and peptides. 

The GlycOCATCH resin is provided in a convenient spin column format. The box contains 4 GlycOCATCH Microspin columns with a capacity to bind 50 µg mucin-type O-glycoprotein each (200 µg total). GlycOCATCH is provided together with 200 units of SialEXO®, for removal of sialic acids, and 200 units of OpeRATOR®, an optional elution method of the bound O-glycoprotein.

Detailed Information

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.

Key Characteristics

  • Binds mucin-type O-glycosylated proteins and peptides
  • 2h binding
  • Desialylation using SialEXO® (included) increases binding
  • Glycoproteins and peptides carrying mucin-type O-glycans

Applications

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Download Poster

Antibody Characterization

Poster from ASMS 2018

"A Novel O-glycoprotease with Applications in O-glycan Analysis using Mass Spectrometry"

How to Order

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Quotations

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Documents

Instructions

 

 

Product Specifications

 

 

Safety Data Sheet

Certificate of Analysis

FAQ and Support

Popular FAQ

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.

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.

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.

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

 

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