Deglycosylation of N-glycosylated proteins using PNGase F
Kaia Kukk
Abstract
The described protocol was used to confirm that NADPH-cytochrome P450 reductase from Helianthus annuus was N- glycosylated when recombinantly expressed in Pichia pastoris ( Komagataella phaffii ).
Steps
12,5 µl of yeast lysate containing sufficient amount of target protein for detecting by Western analysis was pipetted into a tube. Two samples were prepared in parallel, one for negative control without N- glycosidase and the other with PNGase F.
0,5 µl of 10% sodium dodecyl sulfate (SDS) and 1 µl of 1M dithiothreitol (DTT) were added.
The mixtures were incubated at 95 °C for 5 min.
The mixtures were cooled to room temperature after which 2 µl of 0,5 M Tris-HCl, pH 8 and 2 µl of 10% Triton X-100 were added.
2 µl of PNGase F (500 U/ml) was added to one of the samples. Instead of N- glycosidase, water was added to the negative control sample.
The samples were incubated overnight at 37 °C.
SDS-PAGE sample buffer was added and the samples were heated for 5 min at 99 °C. SDS-PAGE and Western analysis were carried out to see whether the molecular weight of the target protein had decreased.
