The Gly-X™ N-Glycan Rapid Release and Labeling with 2-AB Express™ kit utilizes a novel in-solution enzymatic protein deglycosylation followed by on-matrix Express on-matrix labeling of released N-glycans with 2-AB dye, with no prior drydown. With deglycosylation carried out in solution, the method is simple, rapid and suitable for automation. After a simple clean-up step, the 2-AB-labeled glycan samples are ready for analysis by UHPLC, LC-MS, MS/MS and other methods.
Sample prep time: ~2.5 hours.
Other benefits include:
Equipment and reagents provided by user:
Size: 1 kit (24-ct)
Product Code: GX24-2AB
Q. Do you have a list of masses for adduct ions of labeled N-glycans seen by MS in positive and negative mode?
A. Yes, we have a downloadable Excel file listing masses of adduct ions of unlabeled and labeled glycans. The positive mode tab shows glycans labeled by 2-AB, InstantAB, 2-AA, Procainamide and InstantPC. The negative mode tab shows glycans labeled by 2-AB, InstantAB, 2-AA, Procainamide and APTS.
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. You would have to test to ensure that labeled glycans from > 40 µg of your protein can be successfully loaded onto the Gly-X Cleanup Matrix without blockage.
Q. My samples are not loading completely onto the Gly-X Cleanup Matrix. What is happening?
A. This may be caused by the nature of your protein sample, or by matrix effects caused by the composition of your formulation buffer. This can be addressed by using less protein per reaction, or by buffer exchanging your protein prior to starting the Gly-X protocol.
Q: Can I use Eppendorf microtubes for the Denaturation and Digestion Steps, rather than a PCR plate?
A: Yes, leave the tubes open for the heating steps, and ensure that material does not get in the lid during mixing.
Q: Is it possible to use a plate shaker/heater such as the Eppendorf Thermo Mixer C formixing and heating during the Denaturation and Digestion protocols?
A: Yes, this is possible. Mix rate should be high enough to vortex material in wells.
Q: What is the Finishing Reagent for prior to the 2-AB labeling step?
A: The Finishing Reagent is a weak acid, and drives conversion of the glycosylamine (-NH2) to a glycan with a free reducing end (-OH) prior to 2-AB labeling by reductive amination.
Q: For the 2-AB labeling step, the heat block is at 75°C. Is this too high for 2-AB labeling?
A: There is some inefficiency of heat transfer between the block and the plate, so the in-plate temperature is ~65°C. If performing the incubation in an oven, the temperature should be set to 65°C.
Q. Can I use the GX96-2AB 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 GX24-2AB:
|WS0343||Gly-X N-Glycanase (PNGase F)|
|WS0344||Gly-X Digestion Buffer|
|WS0359||Gly-X 2-AB Dye|
|WS0360||Gly-X 2-AB Reductant|
|WS0361||Gly-X 2-AB Catalyst|
|WS0362||Gly-X 2-AB Finishing Reagent|
This kit contains multiple modules, each with their own Certificate of Analysis, which may be searched for & accessed here.