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.

Membrane proteins: Removal of N-Linked Glycans from membrane protein samples using this enzyme has been described by Contessotto et al. Matrix Biol. 2019 (See applications section below)

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A Novel Methanol-Free Platform for Extracellular Expression of rhGM-CSF in Pichia pastoris. Molecular Biotechnology. 2019
Roghayeh Shirvani, Sajjad Yazdanpanah, Mohammad Barshan‑tashnizi*, Maryam Shahali*

TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals. Frontiers Immunology. 2019
José M. Jiménez, Michelle L. Salazar, Sergio Arancibia, Javiera Villar, Fabián Salazar, Gordon D. Brown, Ed C. Lavelle, Luisa Martínez-Pomares, Jafet Ortiz-Quintero, Sergio Lavandero, Augusto Manubens and María Inés Becker*

Distinct glycosylation in membrane proteins within neonatal versus adult myocardial tissue. Matrix Biology. 2019
Contessotto P, Ellis B, Jin C, Karlsson NG, Zorlutuna P, Kilcoyne M, Pandit A*.

Parsimonious Charge Deconvolution for Native Mass Spectrometry. Journal of Proteome Research. 2018
Marshall Bern, Tomislav Caval, Yong J. Kil, Wilfred Tang, Christopher Becker, Eric Carlson, Doron Kletter, K. Ilker Sen, Nicolas Galy, Dominique Hagemans, Vojtech Franc, and Albert J. R. Heck*

Quantitative assessment of mAb Fc glycosylation of CQA importance by capillary electrophoresis. Electrophoresis. 2018
Marton Szigeti , Jeff Chapman , Beata Borza and Andras Guttman*

Methods and apparatuses for determining the intact mass of large molecules from mass spectrographic data. Published Patent Application. 2018
Marshall Bern

Efecto de la eliminación enzimática de N-glúcidos sobre la estructura y propiedades inmunogénicas y antitumorales de las hemocianinas de moluscos en mamíferos. PhD Thesis. 2018
Salazar Luco, Michelle Luna, Becker Contreras, María Inés

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

Deglycosylation of Membrane proteins, as described by Contessotto et al. Matrix Biol. 2019

Deglycosylation of Hemocyanins, as described  by Jimenez et al. Front. Immunol. 2019

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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Size

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