The Glycna Assay system can be applied in these fields, such as deglycosylation, where N-linked glycans, detection and analysis using the LabChip GXII Touch instrument, fluorescent labeling of the released glycans in presence of released proteins and separation by microchip based capillary electrophoresis (CE) to detect the glycosylation patterns of monoclonal antibodies.
Advantages:
1. High efficiency: it can deal with one sample less than 45 seconds, nearly 1.5 hours for 96 samples.
2. The relative amounts of each glycan present can be determined by software.
3. Predictable, reproducible results can be obtained by all-included reagent set.
4. Reagents, which can be offered in 96-well plate, format for convenience and ease of automation.
5. Automatic sampling from a 96-well plate.
Sample preparation:
Digestion
Labeling
Can label 8µL of digested sample?
Sample buffer conditions resulting in optimal glycan peak intensity can be detected while labeling 8µL of digested sample. More or less will result in lower signal intensity.
Break point
Sealing and storing the dried samples at -20°C after labeling and reconstituting the next day.
Mixing
Using a plate shaker with max speed is highly recommended.
Incubators:
Inspect samples carefully to make sure that they are totally dry. Using an incubator or oven will increase the drying time to more than 2 hours.
Reconstitution
These are some basic information about the glycan assay workflow.
Advantages:
1. High efficiency: it can deal with one sample less than 45 seconds, nearly 1.5 hours for 96 samples.
2. The relative amounts of each glycan present can be determined by software.
3. Predictable, reproducible results can be obtained by all-included reagent set.
4. Reagents, which can be offered in 96-well plate, format for convenience and ease of automation.
5. Automatic sampling from a 96-well plate.
Sample preparation:
- Spin-down: all plates should be spun down before removing the seal to collect any reagent, which then might be attached to the plate seal.
- Cutting the plates: The 96-well Denaturing, PNGase- F, and Dye Plates should be cut vertically into sections of 24, 48, or 72 wells if running fewer than 72 samples.
- Storage: freezing the remaider after cutting the plate.
- Partial plates: stacking the plate on top of a 96-well PCR plate is very helpful to support.
- Plate seals: using plate seals with a minimum thermal range of -20°C to 80°C, a rubber roller is recommended for all plate sealing.
Digestion
- Samples after digestion can be frozen at -20°C over a night.
- Incubators: incubators or ovens can not be used for incubation. If an incubator is applied in the labeling step, the acetic acid fumes should be processed carefully, while opening the incubator door.
Labeling
Can label 8µL of digested sample?
Sample buffer conditions resulting in optimal glycan peak intensity can be detected while labeling 8µL of digested sample. More or less will result in lower signal intensity.
Break point
Sealing and storing the dried samples at -20°C after labeling and reconstituting the next day.
Mixing
Using a plate shaker with max speed is highly recommended.
Incubators:
Inspect samples carefully to make sure that they are totally dry. Using an incubator or oven will increase the drying time to more than 2 hours.
Reconstitution
- Add 100µL of water to dried samples.
- Seal plate with an adhesive plate seal.
- Mix samples on a plate shaker at least 1 minute, making sure that the pellet has completely dissolved.
- Spin plate at 1200g for 1 minute.
- Remove the plate seal, and prepare the plate to run on labchip GXII Touch.
These are some basic information about the glycan assay workflow.