PNGase-F-3D

PNGase F (CarboClip)

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Introduction: CarboClip is a recombinant peptide-N-Glycosidase F (PNGase F), from Elizabethkingia sp, overexpressed in E. coli that contains a hexahistidine tag in its C-term and cleaves between the GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins. CarboClip is provided glycerol-free as a lyophilized powder containing Tris-HCl, NaCl2, and Na2EDTA.

Activity: After reconstitution in 150 μL of UltraPure H2O, 1 μl of CarboClip can deglycosylate > 95% of ribonuclease B (1 mg) in less than 20 minutes. Activity is calculated on a 12 % SDS-PAGE and percent of deglycosylation using densitometry.

Storage: Lyophilized powder: -80 °C and -20 °C >1 year. At room temperature 10 days. Reconstituted protein should be stored at 4 °C and is stable for 2-3 months.

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Product Description

Removal of N-Linked Glycans from Glycoproteins, Microwave deglycosylation, Proteomics, Glycomics…

Booklet CarboClip 500 mU

Booklet CarboClip 2500 mU

 

Denaturing deglycosylation of 100 µg of glycoproteins and analysis

  1. Add 100 µg of glycoprotein and fill with UltraPure H2O (milliQ, NanoPure, etc) until 70 µl.
  2. Add 10 µl of Denaturing Buffer (5% SDS, 400 mM DTT).
  3. Incubate at 100°C for 10 minutes.
  4. Add 10 µl of reaction buffer (500 mM Sodium Phosphate (pH 7.5)) and 10 µl of 10% NP-40.
  5. Add 0.1 µl of Carboclip PNGase F
  6. Incubate at 37°C for 30 min.
    (Depending on the glycoprotein up to overnight incubation could be necessary)
  7. 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

 

  1. Add 2 µl of PNGase F (CarboClip) to 100 µl of IgG (10 µg/µl) diluted in nanoPure H2O.
  2. Incubate at 60 ºC for 2h.
  3. Clean and concentrate the sample using an amicon device
  4. Add 1 µl of concentrated IgG to the polished steel MALDI-TOF plate,
  5. Add 1 µl of SuperDHB (diluted in ACN at 20 µg/µl).
  6. Let it dry and analyze by MALDI-TOF
  7.  Flex control-ultraflex tof-tof software
    Method: LP_30-210_KDa.par.
    Set laser Attenuator “3_medium”.
    Mass range 68,000 – 170,000.

See  full protocol: IgG deglycosylation analysis using MALDI-TOF

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Additional Information

Size

PNGase F- 100 IUB mU, PNGase F- 500 IUB mU, PNGase F- 2,500 IUB mU, PNGase F- 5,000 IUB mU