Recombinant PNGase F Kit (CAT#: CB-P248-K) Datasheet

Product Type
Kit
Description
PNGase F is a cloned glycosidase, cloned from the microorganism of Queen Elizabeth and expressed in E. coli. The enzyme does not contain glycerol (for best performance in HPLC enhancement methods), as well as reaction buffer, denaturing solution and NP-40 solution, which can achieve effective deglycosylation. PNGase F is suitable for releasing all types (high mannose, hybrid and complex) N-linked glycans from glycoproteins and glycopeptides. PNGase F does not remove the oligosaccharides containing α(1-3) linked core fucose that are usually found on plant glycoproteins.
Applications
Peptide N-glycosidase F (PNGase F) is suitable for release of N-linked glycans in solution, and from immobilized samples. The enzyme cleaves between the innermost GlcNAc of the oligosaccharide moiety at its attachment point to the asparagine residue on the protein and subsequently converts the asparagine into aspartic acid. Released glycans with free reducing terminus can be labelled using CreaTag labelling technology for fluorescence and high MS sensitivity detection.
Size
150 µL
Sample
Glycoproteins and glycopeptides containing N-linked glycans
Sample Volume
Kit contains 75,000 units of PNGase F at concentration of 500,000 units/ml. Sufficient for approximately 150 samples. As a guideline up to 100 µg of glycoprotein per sample.
Species Reactivity
PNGase F is suitable for release of all types (high-mannose, hybrid and complex) N-glycans from glycoproteins and glycopeptides. Xaa-Asn-Xaa sequence is the minimal peptide substrate for this enzyme. Note that some non-mammalian glycans from sources such as plants, insects and parasites carrying α1-3 linked core fucose will not be cleaved with PNGase F. For these samples PNGase A can be used.
Components
PNGase F (Elizabethkingia miricola) suuplied in 50 mM NaCl 5 mM EDTA 20 mM Tris-HCl pH 7.5 - 1 vial of 0.15mL
10X Reaction Buffer 500 mM sodium phosphate (pH 7.5 at 1X dilution) - 1 vial of 1.0 mL
10X Denaturation Solution 5% SDS 400 mM DTT - 1 vial of 1.0 mL
NP-40 10% solution - 1 vial of 1.0 mL
Handling Advice
Denaturing reaction conditions:
1. Make up sample volume to 9 µL with ultrapure water.
2. Add 1 µL of 10X Denaturation Solution to each glycoprotein sample. Close the reaction vials, vortex thoroughly and briefly centrifuge to ensure the samples are completely dissolved.
3. Incubate the samples at 100°C for 10 minutes.
4. Add 2 µL of 10X Reaction Buffer to each glycoprotein sample.
5. Add 2 µL of 10% NP-40 solution.
6. Adjust the reaction volume to 20 µL by adding 6 µL of water.
7. Add 1 µL of PNGase F. Close the reaction vials, mix gently and briefly centrifuge.
8. Incubate the samples at 37°C for 1h.
Storage
Store at 4°C. Protect from sources of heat and light
Note: PNGase F is inactivated after 10 minutes at 75°C.
Shelf Life
12 months
Research Use
For research use only. Not for human or drug use

All products and services are for Research Use Only. Do Not use in humans.

ONLINE INQUIRY

Creative Biolabs has established a team of customer support scientists ready to discuss ADCC/CDC optimization strategies, antibody production, bioinformatics analysis and other molecular biology/biotechnology issues.

  • *
  • *
  • *
USA

UK