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

150 µL

Glycoproteins and glycopeptides containing N-linked glycans

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.

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.

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

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.

Store at 4°C. Protect from sources of heat and light
Note: PNGase F is inactivated after 10 minutes at 75°C.

12 months

For research use only. Not for human or drug use