EB10 Cartridges
Specifications for EB10 Cartridges
Application: For purification of glycans from a variety of complex mixtures (including removal of salts and detergents).
Description: The cartridges contain a unique non-porous Electronic Interaction (EI) matrix. This acts like a super-hydrophobic resin that binds even very hydrophilic glycans. Most salts and detergents either simply pass through the cartridges or bind very lightly and can be washed off before the glycans are eluted.
Binding capacity: Each EB10 cartridge can typically bind up to 50 μg of O- or N-linked glycans.
Structural Integrity: No detectable (< 2 mole per cent) loss of sialic acid, fucose, sulfate, or phosphate.
Number of Samples: EB10 cartridges are designed for single use only.
Suitable Samples: A wide range of glycans can be purified. These include N-linked and O-linked type oligosaccharides, tri-saccharides and larger structures. The cartridges are not suitable either for monosaccharides or disaccharides which are generally bound too weakly for efficient purification or for large linear poly-sialylated glycans which can be bound very tightly to the resin.
Glycan samples must be applied to the cartridges in solutions that are substantially aqueous.
Binding efficiency: > 95 % for most glycans
Binding Selectivity: Essentially stoichiometric binding and elution for most complex glycan mixtures.
Storage: Store at room temperature in the dark. Protect from sources of heat, light, and moisture. The cartridges are stable for at least two years as supplied.
Shipping: The product can be shipped at ambient temperature.
Handling: Ensure that any glass, plasticware or solvents used are free of glycosidases and environmental carbohydrates. Use powder-free gloves for all sample handling procedures and avoid contamination with environmental carbohydrate.
Safety: Please read the Material Safety Data Sheets (MSDS's) for all chemicals used.
All processes involving hazardous reagents should be performed using appropriate personal safety protection - eyeglasses, chemically resistant gloves (e.g. nitrile), etc. - and where appropriate in a laboratory fume cupboard.
For research use only. Not for human or drug use.
Additional Reagents and Equipment Required
• Pure water (HPLC grade)
• Methanol
• 1M sodium hydroxide (aq)
• 30% acetic acid (aq)
• Acetonitrile (HPLC grade)
• Trifluoroacetic acid (Analar grade)
• Wash A: 5 % (v/v) acetonitrile (aq) plus 0.1% trifluoroacetic acid
• Wash B: 50 % (v/v) acetonitrile (aq) plus 0.1% trifluoroacetic acid
• Pipettes
• 0.5 μm or 0.2 μm microcentrifuge filters
• Microcentrifuge
Introduction
EB10 cartridges have been designed for purification of glycans from non-carbohydrate
material including salts, proteins, and detergents. Applications include cleanup of glycans following
hydrazinolysis, endoglycosidase digests (including PNGase F digests), and enzyme treatment, and before
and after fluorescent labeling.
Outline of EB10 Cleanup Protocol
1)Wash the cartridge
The EB10 cartridge is washed with successive washes of water, 1 M sodium hydroxide, water, 30% acetic acid, then water.
2)Prime the resin
The active surface of the resin in the carrtidge is primed by washing with acidic aqueous solutions of acetonitrile.
3)Prepare the glycan sample
Dilute out any organic solvents and remove viscous or particulate material from the sample.
4)Apply the glycan sample
The aqueous solution of glycan sample is applied to the cartridge.
5)Wash off the non-glycan contaminants
Non-glycan contaminants such as salts and detergents are washed out using dilute acidic aqueous acetonitrile.
6)Elute the glycans
Bound glycans are washed off the cartridge using a higher concentration of acidic aqueous acetonitrile.
7)Post elution workup
The eluted glycan solution is filtered.
8)Analyse the glycans
The glycans are now ready for analysis.
Time Line for Cleanup
The EB10 glycan cleanup procedure typically takes around 65 minutes :
Procedure | Time | Elapsed Time (minutes) |
Filter samples | 20 min | 20 |
Wash and prime cartridges | 15 min | 25 |
Apply sample | 10 min | 35 |
Wash off non-glycan contaminants | 15 min | 50 |
Elute glycans | 15 min | 65 |
Preparation of Cartridges
1) Wash the cartridge
Prepare each EB10 cartridge by washing with the following:
Reagent | Volume (ml) |
methanol | 0.5 |
1M sodium hydroxide | 0.5 |
water | 1 |
30% acetic acid | 1 |
water | 1 |
This removes any impurities that may have bound to the resin matrix during storage.
If the flow is restricted, e.g. by an air gap, then apply a slight pressure to the top of the cartridge (e.g. using a clean, gloved thumb) in order to resume normal flow.
N.B. Do not force liquid through the resin bed either by applying excessive pressure or by using a vacuum as these can cause blockage of the porous frits or leakage of resin through the bottom frit.
2)Prime the cartridge
Prime each cartridge with the following:
Reagent Volume (ml)
WashB 0.5
Wash A 1
This prepares the surface of the resin for binding of the glycans.
3)Prepare the glycan sample
The sample to be cleaned must be in either an aqueous buffer or one containing only a low percentage of organic solvent. If the sample contains organic solvent then dilute with water until the organic solvent content is less than 5 % by volume.
Some types of sample may contain particulate or viscous material that can block the flow through the EB10 cartridges. These include some glycoproteins subjected to glycan release by endoglycosidase treatment. Monoclonal antibodies treated with PNGase F are particularly prone to formation of viscous material. In such cases, to minimize blockage of the EB10 cleanup cartridges:
a. Dilute each sample with 500 μl water.
b. Mix thoroughly by vortexing.
c. Spin in a micro-centrifuge (typical conditions are centrifugation at 10,000 rpm for 15 minutes).
c. Carefully pipette out the supernatant and apply to the prepared EB10 cartridge (see step 4).
4)Apply the sample
Apply the sample to the cartridge.
Glycans should bind to the matrix while salts and non-hydrophobic non-glycan contaminants pass through.
5)Wash off non-glycan contaminants
Wash the cartridge with the following:
Reagent Volume (ml)
water 0.7
Wash A 0.7
This removes residual salts and non-hydrophobic non-glycan material from the column.
Discard these washes into a suitable waste container.
6)Elute the glycans
Place the cartridge over a collection vessel and recover the glycans by eluting with 4 x 0.2 ml of Wash B allowing each aliquot to drain before the next is applied.
Glycans should be eluted while hydrophobic material such as certain peptides, proteins, and detergents remain bound to the solid phase matrix.
7)Dry the eluted glycans (optional)
If appropriate, evaporate the glycan containing fraction to dryness, then redissolve in a desired volume of water or solvent for further analysis.
8)Filter the eluted glycans
Samples should be filtered to at least 0.5 μm.
We recommend that filtration is performed using microcentrifuge spin filters with an inert membrane – e.g. PTFE or polypropylene.