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  • Remove-iT® PNGase F

    Description

    Remove-iT® PNGase F is an amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (1). Remove-iT PNGase F is tagged with a chitin binding domain (CBD) for easy removal from a reaction and is supplied glycerol free for optimal performance in HPLC and MS intensive methods.

    Product Source

    Remove-iT PNGase F is purified from Flavobacterium meningosepticum (2).

    Specificity

    P0706

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    DTT-2010X
    GlycoBuffer 2-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 5 μg of DTT denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 μl.

    5 µg of RNase B are denatured with 1X DTT at 55°C for 10 minutes. After the addition of 1X GlycoBuffer 2, two-fold dilutions of Remove-iT PNGase F are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE.

    Reaction Conditions
    1. Combine 10–20 μg of glycoprotein, 1 μl of 10X DTT and H20 (if necessary, to make a 10 μl total reaction volume).
    2. Denature glycoprotein by heating reaction at 55°C for 10 minutes.
    3. Make a total reaction volume of 20 μl by adding 2 μl 10X GlycoBuffer 2, H20 and 1–5 μl Remove-iT PNGase F.
    4. Incubate reaction at 37°C for 1 hour.

    Storage Temperature

    4°C

    Storage Conditions

    20 mM Tris-HCl
    50 mM NaCl
    5 mM Na2EDTA
    pH 7.5 @ 25°C

    Heat Inactivation

    75°C for 10 min

    Molecular Weight

    Apparent: 41000 daltons

    Notes

    1. To deglycosylate a native glycoprotein, longer incubation time, as well as more enzyme, may be required.
    2. Using typical RNase B denaturing conditions with NEB Glycoprotein Denaturing Buffer, containing SDS and DTT, Remove-iT PNGase F yields a higher concentration of 500,000 U/ml.
    3. If using Remove-iT PNGase F under typical PNGase F denaturing conditions, it is essential to have NP-40 in the reaction mixture as Remove-iT PNGase F is inhibited by SDS. It is not known why this non-ionic detergent counteracts the SDS inhibition
    4. Remove-iT PNGase F will not cleave N-linked glycans containing core α1-3 Fucose.
    5. Recommended storage temperature is 4°C, avoid repeat freeze-thaw cycles
    6. Removal of Remove-iT PNGase F from the deglycosylation reaction can be scaled up linearly with larger volumes of chitin magnetic beads.
    7. Chitin Magnetic Beads Binding Capacity is 0.4 µg/µl of CBD-tagged protein.
    8. To remove 1-5 µl of Remove-iT PNGase F use 50 µl of chitin magnetic beads.

    References

    1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
    2. Plummer, T. H., Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.

    FAQs

    1. What is the tag on Remove-iT® PNGase F?
    2. I tried deglycosylating my glycoprotein with Remove-iT® PNGase F but did not see removal of the carbohydrate. What could be the problem?
    3. What is the difference between PNGase F and Remove-iT® PNGase F?
    4. What is the difference between Remove-iT® PNGase F and Endo H?
    5. How much Remove-iT® PNGase F should I use to remove my carbohydrate under native or DTT denaturing conditions?
    6. Does Remove-iT® PNGase F work in Urea?
    7. What are the typical reaction conditions for Remove-iT® PNGase F?
    8. How do I eliminate Remove-iT® PNGase F from a reaction?
    9. What is the binding capacity of the Magnetic Chitin Beads used to eliminate Remove-iT® PNGase F?
    10. Is Remove-iT® PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
    11. Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers?
    12. What is a good endoglycosidase substrate?
    13. Do detergents inhibit exoglycosidases/endoglycosidases?
    14. What are glycosidases and their uses?

    Protocols

    1. Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
    2. Reaction Conditions for Remove-iT® PNGase F (P0706)

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Application Notes

    Citations

    • Zhao H, Blazanovic K, Choi Y, Bailey-Kellogg C, Griswold KE (2014). Gene and protein sequence optimization for high-level production of fully active and aglycosylated lysostaphin in Pichia pastoris Appl Environ Microbiol. 80(9), 2746-53. PubMedID: 24561590, DOI: 10.1128/AEM.03914-13

    Quality Control

    Quality Assurance Statement

    • No contaminating exoglycosidase or Endoglycosidase F1, F2 or F3 activity could be detected. < 0.01% of Endoglycosidase F1 could be detected. No contaminating proteolytic activity could be detected.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Glycosidase Activity (TLC):
      The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide.  The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
    • Protease Activity (SDS-PAGE):
      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.