The Gly-X™ N-Glycan Rapid Release and Labeling with InstantQ™ kit utilizes a novel in-solution enzymatic protein deglycosylation followed by rapid labeling of released N-glycans with InstantQ dye. After a simple clean-up step, the glycan samples are ready for analysis on the Gly-Q Glycan Analysis System. With deglycosylation and labeling carried out in solution, the method is simple, rapid and suitable for automation.
Sample prep time: ~1 hour.
Other benefits include:
Equipment and reagents provided by user:
Size: 1 kit (96-ct)
Product Code: GX96-IQ
Gly-X with InstantQ Kit
|Sample prep time||96 samples in 1 hour|
|Protein load range||1-40 µg|
|Label||Proprietary instant label|
|Sialylated species||>95% retained|
|Sample stability||4°C for up to 5 days|
|Sample stability||-20°C for up to 3 months|
Kits and Modules
|GX96-IQ||Gly-X with InstantQ Kit (96-ct)|
|GX96-302IQ||Gly-X with InstantQ Deglycosylation and Labeling Module Set (96-ct)|
|GX24-302IQ||Gly-X with InstantQ Deglycosylation and Labeling Module Set (24-ct)|
|GX96-301||Gly-X InstantQ Labeling Module|
|GX96-302||Gly-X InstantQ Cleanup Module|
|G5524-60010 KIT||AssayMAP PA50 protein A affinity purification kit (96-ct)|
|GQ103||Gly-Q Cartridge Module|
Standards and Controls
|GKSQ-500||Gly-Q Migration Standards (upper and lower)|
|GKSQ-503||Gly-Q GU Ladder|
|GKSQ-505||Gly-Q Alignment Standards Set (GKSQ-500 & GKSQ-503)|
|GKSQ-005||Human IgG N-Glycan Library|
|GKSQ-009||InstantQ Labeled RNase B Library|
|GKSQ-301||InstantQ Labeled G0|
|GKSQ-302||InstantQ Labeled G0F|
|GKSQ-317||InstantQ Labeled G1|
|GKSQ-316||InstantQ Labeled G1F|
|GKSQ-304||InstantQ Labeled G2|
|GKSQ-305||InstantQ Labeled G2F|
|GKSQ-315||InstantQ Labeled A1F|
|GKSQ-312||InstantQ Labeled A2|
|GKSQ-313||InstantQ Labeled A2F|
|GKSQ-103||InstantQ Labeled Man5|
|GKSQ-104||InstantQ Labeled Man6|
|GKSQ-105||InstantQ Labeled Man7|
|GKSQ-106||InstantQ Labeled Man8|
|GKSQ-107||InstantQ Labeled Man9|
Q: When do I need to use the Gly-X Blocker with my deglycosylation reaction? What does the Blocker do?
A: The Gly-X Blocker is optional. It is not required for most biotherapeutics e.g. IgGs, Fc fusion proteins. However, if you observe an uneven/rounded baseline during analysis we suggest running the protocol with and without the Blocker to test whether it is required. Potential scenarios that may require the Blocker are:
a) if your glycoprotein is <50 kDa;
b) if the glycoprotein contains a large proportion of basic amino acids;
c) if your sample is a complex matrix such as a biofluid.
The Blocker ensures that labeled protein does not interfere with N- glycan analysis by blocking free amine groups on the glycoprotein before they can be labeled with InstantQ dye.
Using the blocker may cause the reaction to turn yellow, particularly when there is a lot of protein material. This is caused by Maillard reaction products and is not a cause for concern. Please contact ProZyme for further details.
Q. Why should I use 0.1% formic acid to elute samples from Protein A ready for Gly-X with InstantQ, rather than a low pH glycine solution?
A. Glycine has a primary amine which will react with the InstantQ label, so we suggest that 0.1% formic acid is used as a protein eluent. Alternatively, glycine may be used but samples must be desalted before deglycosylation.
Q. Can I use ProZyme AssayMAP Protein A cartridges to purify proteins ready for Gly-X with InstantQ, and elute with 0.1% formic acid?
Q. What is the lower limit of glycoprotein I can use with the Gly-X/InstantQ protocol?
A. Each laboratory will need to establish the lower limit for each specific protein. 0.125 mg/ml can be used as a starting point.
Q: Can I use more than the recommended upper limit of 40 µg protein per reaction?
A: Maybe, although it will depend on the protein. When using >40 µg protein the user should check that relative percent area data maintains linearity.
Q: Can I run glycans labeled with InstantQ dye on other CE systems?
A: The LIF detector typically used on the Beckman PA800-plus for APTS glycans is not compatible with the InstantQ Dye excitation wavelength. Suitability on other systems may be determined by the user.
Q. Can I use the GX96-IQ kit with a Waters vacuum manifold?
A. Yes, if GX200 Gly-X Vacuum Manifold Spacer (Waters Manifold) is used in place of GX100 during the Elute step. GX200 provides the correct distance between the Cleanup Place and Collection Plate when using a Waters vacuum manifold (#186001831). This is critical to avoid crosstalk when eluting labeled N-glycans into the Collection Plate. Please contact ProZyme at firstname.lastname@example.org for further details.
Product Safety Documentation for GX96-IQ:
|WS0343||Gly-X N-Glycanase (PNGase F)|||
|WS0344||Gly-X Digestion Buffer|
|WS0260-GP||InstantQ Dye Solvent|
|WS0354||InstantQ Activation Reagent|
This kit contains multiple modules, each with their own Certificate of Analysis, which may be searched for & accessed here.