How to use Rapid PNGase F to release N-glycans from antibodies
The following deglycosylation protocol is recommended for use with New England BioLabs Rapid PNGase F. Note that either heat blocks, as shown in this video, or a thermocycler 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 the pipette. Incubate both tubes at 80 degrees Celsius for two minutes. Cool to room temperature. 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 1.25 molar 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 Reaction
Load 15 microliters each of the test sample and negative control side by side in 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. De-stain the gel with water solution following the manufacturer's protocol. Take a picture of the de-stained gel on a white 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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