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PNGase F (Glycerol Free) > FAQ |  | PNGase F (Glycerol Free) FAQ
See the Protein Tools FAQ also.
Q1: I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
Q2: What is the difference between PNGase F and Endo H?
Q3: How much PNGase F should I use to remove my carbohydrate under native conditions?
Q4: How do I inhibit PNGase F?
Q5: What is a good endoglycosidase substrate?
Q6: Does PNGase F work in Urea?
Q7: Do detergents inhibit exoglycosidases/endoglycosidases?
Q8: What are the typical reaction conditions for PNGase F (Glycerol Free)?
Q9: Why is my immunoprecipitated (IP) protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein?
Q10: What are Glycosidases and their uses?
Q1: I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
A1: Do you know how the carbohydrate is attached to the protein? Is it N-, or O-linked (linked to an Asn, or a Ser/Thr)? PNGase F will only remove carbohydrates attached to an Asn. An O-Glycosidase may help for O-linked carbohydrates but we do not carry this type of endoglycosidase. (Try Prozyme, Inc. as a source for O-Glycosidase.) If you are certain you have N-linked carbohydrates, make sure that you denature the protein prior to deglycosylation. The secondary and tertiary structure of proteins can prevent endoglycosidases from reaching their substrate, thus making denaturation a crucial step in efficient cleavage. If you do not want to denature then consider adding more enzyme and longer incubation times. If you have denatured the protein, be certain to add NP-40 (to protect PNGase F from the SDS in the denaturation step). Outside these reaction conditions there is the possibility that the carbohydrate may be resistant to PNGase F (a rare occurrence). This happens when the core N-acetyl-glucosamine is modified by an alpha1-3Fucose (often found in plant proteins).
Q2: What is the difference between PNGase F and Endo H?
A2: PNGase F removes all types of N-linked (Asn linked) glycosylation; high mannose, hybrid, bi, tri, and tetra antennary. You will choose this enzyme if your goal is to remove all N- linked carbohydrates without regard to type. Endo H removes only high mannose and some hybrid types of N-linked carbohydrates. You would choose this enzyme to more closely determine the type of N-linked glycosylation, or if you know that the protein has a carbohydrate sensitive to Endo H.
Q3: How much PNGase F should I use to remove my carbohydrate under native conditions?
A3: When the protein is not denatured PNGase F can have a difficult time reaching the cleavage site of the carbohydrate (because of the secondary and tertiary protein structure). Sometimes extra enzyme and extended incubation times can help but these values are specific to each protein and must be determined empirically.
Q4: How do I inhibit PNGase F?
A4: SDS is an excellent inhibitor of PNGase F.
Q5: What is a good endoglycosidase substrate?
A5: We suggest RNase B (NEB# P7817S)
Q6: Does PNGase F work in Urea?
A6: PNGase F is stable in 2.5M urea at 37C for 24 h and still possesses 40% of its activity in 5 M urea1.
1. Maley, F., et al, Anal Biochem, 180, 195-204 (1989)
Q7: Do detergents inhibit exoglycosidases/endoglycosidases?
A7: At moderate levels (0.5-1.0% ionic and non-ionic detergents) most of the glycosidases show satisfactory activity or are unaffected. One major exception is PNGase F as it is inhibited by SDS.
Q8: What are the typical reaction conditions for PNGase F (Glycerol Free)?
A8: Typical reaction conditions are as follows:
1. Combine 1-20 µg of glycoprotein, 1 µl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 µl total reaction volume.
2. Denature glycoprotein by heating reaction at 100°C for 10 minutes.
3. Make a total reaction volume of 20 µl by adding 2 µl 10X G7 Reaction Buffer, 2 µl 10% NP40, H2O and 1-5 µl PNGaseF.
4. Incubate reaction at 37°C for 1 hour.
Note: Reactions may be scaled-up linearly to accommodate larger reaction volumes.
Q9: Why is my immunoprecipitated (IP) protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein?
A9: Proteases are common contaminants in IP reactions. When a protein is denatured it is more susceptible to cleavage by proteases. A protease cocktail containing the following:
Aprotinin 10 ug/ml final concentration (dissolve in water)
Benzamidine 1mM final concentration
Pepstatin 10 ug/ml final concentration
Leupeptin 1mM final concentration
EGTA 1 mM final concentration
EDTA 1 mM final concentration )
Make a 1000X concentrated stock of each inhibitor in water; except pepstatin is dissolved in methanol.
Note: PMSF should not be used with PNGase F.
Q10: What are Glycosidases and their uses?
A10: Glycosidases are used to get information about the carbohydrate groups attached to glycoproteins and glycopeptides. They come in two varieties, endoglycosidases that cleave entire carbohydrate groups from proteins and exoglycosidases that remove monosaccharides from the nonreduced ends of the carbohydrate. A reduced end is one generated by an endoglycosidase. Researchers frequently use an endoglycosidase followed by one or more exoglycosidases and then analyze the products using SDS-PAGE or various types of liquid chromatography.
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