PNGase F-Fast

One-step procedure
1. Dissolve 100 μg of antibody or glycoprotein in deionized water and dilute to 16 μL with deionized water;
2. Add 4 μL of reaction buffer (0.25 M sodium phosphate pH 7.50, 0.1 M DTT (HY-15917), 25 g/L Lauric acid (HY-Y0366) and gently pipette to mix.
3. Add 1 μL of PNGase F-Fast and gently pipette to mix.
4. Incubate at 50°C for 10 min.
5. After derivatization, N-glycans can be directly isolated and purified using solid-phase extraction for downstream glycoform analysis.

Two-Step Procedure
For some glycosylated antibodies, complete deglycosylation requires sample preheating. The steps are as follows:
1. Dissolve 100 μg of antibody or glycoprotein in deionized water and adjust the volume to 16 μL with deionized water;
2. Add 4 μL of reaction buffer (same composition as above) and gently pipette to mix;
3. Incubate at 80°C for 2 minutes, then cool on ice;
4. Add 1 μL of PNGase F-Fast and gently pipette to mix;
5. Incubate at 50°C for 10 minutes;
6. After derivatization, N-glycans can be directly isolated and purified using solid-phase extraction for downstream glycoform analysis.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.