One-step protocol for deglycosylation with Rapid PNGase F

How to use Rapid PNGase F to release N-glycans from antibodies

The following deglycosylation protocol is recommended for use with New England BioLab's Rapid PNGase F.

If a complete shift of the glycoprotein is not observed on SDS page with the test sample treated with Rapid PNGase F using the one-step protocol, it is recommended that a two-step protocol be tried, as some antibodies require a preheating step for efficient deglycosylation. Note that either heat blocks, as shown in this video, or a thermo-cycler, may be used with this protocol.

1/5 Set up Reaction

Label two 1.5 milliliter tubes, one for the negative control and one for the test. Dissolve up to 100 micrograms of IgG in water to a total volume of 16 microliters in both tubes. Add four microliters of 5x Rapid PNGase F buffer to both tubes containing the IgG and water mixture and mix gently with a pipette. Add one microliter of Rapid PNGase F to the tube labeled "Test" and mix gently.

2/5 Start Reaction

Incubate both tubes for 10 minutes at 50 degrees Celsius.

3/5 Prepare to Analyze Reaction

While the reaction is running, prepare 3X reducing SDS loading buffer by mixing together four microliters of 1.25M DTT and 130 microliters of 3X SDS loading buffer. After the Rapid PNGase F reaction is finished, add 10 microliters of 3X reducing SDS loading buffer to each tube. Place the tubes in the heat block and incubate at 95 degrees Celsius for five minutes. Allow the reaction to cool down.

4/5 Analyze the Reaction

Load 15 microliters each of the test sample and negative control side by side, and three microliters of the color pre-stained protein standard on a 10 to 20% Tris-Glycine gel. Run the gel at 130 to 200 volts.

5/5 Visualize Results

Remove the gel from the gel box after the dye has migrated to the bottom. Place the gel in a plastic tray and stain with Simply Blue Stain Solution or any stain of choice, following the manufacturer's protocol.

Destain the gel with water solution following the manufacturer's protocol. Take a picture of the destained gel on a while light transilluminator. Observe how the test IgG treated with Rapid PNGase F migrates to a lower position on the gel, compared to the negative control. If using standard PNGase F from another supplier, we suggest following their recommended protocol for optimum protein deglycosylation.

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