PNGase F Protocol (Denaturing Conditions)

Abstract

PNGase F is the most effective enzymatic method for removing almost all N -linked oligosaccharides from glycoproteins. PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides.

This is a generic PNGase F protocol for denaturing reaction conditions . It is appropriate for both P0704 and P0708.

Before start

Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are as follows:

Steps

Denaturing Reaction Conditions:

Combine 1µg - 20µg, 1µL and H₂O (if necessary) to make a total reaction volume 10µL total reaction volume.

Denature glycoprotein by heating reaction at 100°C for 0h 10m 0s.

Chill denatured glycoprotein 37On ice and centrifuge 0h 0m 10s.

Make a total reaction volume of 20µL by adding: 2µL, 2µL and 6µL.

Note

PNGase F is inhibited by SDS, therefore it is essential to have NP-40 in the reaction mixture under denaturing conditions. Failure to include NP-40 into the denaturing protocol will result in loss of enzymatic activity.

Add 1µL and mix gently.

Incubate reaction at 37°C for 1h 0m 0s.

Analyze by method of choice.

Note

Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.

Abstract

Before start

Steps

Denaturing Reaction Conditions:

推荐阅读