Removal of N-Linked Glycans from Glycoproteins, Microwave deglycosylation, Proteomics, Glycomics…
Denaturing deglycosylation of 100 µg of glycoproteins and analysis
- Add 100 µg of glycoprotein and fill with UltraPure H2O (milliQ, NanoPure, etc) until 70 µl.
- Add 10 µl of Denaturing Buffer (5% SDS, 400 mM DTT).
- Incubate at 100°C for 10 minutes.
- Add 10 µl of reaction buffer (500 mM Sodium Phosphate (pH 7.5)) and 10 µl of 10% NP-40.
- Add 0.1 µl of Carboclip PNGase F
- Incubate at 37°C for 30 min.
(Depending on the glycoprotein up to overnight incubation could be necessary)
- Analyze by SDS-PAGE
Non-denaturing deglycosylation of IgG:MALDI-TOF compatible application.
1. IgG desalting using an Amicon Ultra-0.5 mL Centrifugal Filter device MWCO 30 kDa:
Add your IgG sample to the filter device. If volume is less than 400 µL, add ultra pure water up to 400 µL and spin the sample for 5 minutes at 9,000x g at room temperature. Discard the flow-through. Repeat for a total of three times.
Desalting of antibody is recommended as the presence of salts and detergents could affect the MALDI-TOF-MS acquisition.
2. Antibody Deglycosylation:
Add 3-6 µL of reconstituted CarboClip enzyme for each 50-200 µg of IgG to deglycosylate. Mix the sample by flicking (do not vortex or pipette up and down). Incubate the mixture at 60°C for 1 hour with gentle shaking.
For efficient deglycosylation of the Fab region IgG denaturation could be necessary.
Analysis of protein deglycosylation by MALDI-TOF.
e.g. Deglycosylation of IgG under non-denaturing conditions
- Add 2 µl of PNGase F (CarboClip) to 100 µl of IgG (10 µg/µl) diluted in nanoPure H2O.
- Incubate at 60 ºC for 2h.
- Clean and concentrate the sample using an amicon device
- Add 1 µl of concentrated IgG to the polished steel MALDI-TOF plate,
- Add 1 µl of SuperDHB (diluted in ACN at 20 µg/µl).
- Let it dry and analyze by MALDI-TOF
- Flex control-ultraflex tof-tof software
Set laser Attenuator “3_medium”.
Mass range 68,000 – 170,000.
See full protocol: IgG deglycosylation analysis using MALDI-TOF