PNGase F Protocol (Non-Denaturing Reaction 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 non-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

Non-Denaturing Reaction Conditions:

Note

When deglycosylating a native glycoprotein it is recommended that an aliquot of the glycoprotein is subjected to the denaturing protocol to provide a positive control for the fully deglycosylated protein. The non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.

Combine 1µg - 20µg, 2µL and H₂O (if necessary) to make a total reaction volume 20µL total reaction volume.

Add 2µL - 5µL, mix gently.

Incubate reaction at 37°C for 4h 0m 0s - 24h 0m 0s.

Note

Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.

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

Non-Denaturing Reaction Conditions:

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