Phastgel ief

Sodium dodecyl sulfate-polyacrylamide (10% (w/v)) gel electrophoresis
(SDS-PAGE) was performed as described by Laemmli.^{33 (link)} A NuPAGE Novex System (ThermoFisher Scientific, Bleiswijk,
The Netherlands) with 10% (w/v) Bis-Tris gels was used. Prior to electrophoresis,
all samples were heated for 10 min at 70 °C in NuPAGE LDS sample
buffer with NuPAGE sample reducing agent, according to the instructions
of the manufacturer. Gels were stained with SimplyBlue SafeStain according
to the recommendation of the supplier (ThermoFisher Scientific). For
detection of glycosylated proteins, the SDS-PAGE gel was stained using
periodic acid-Schiff staining (PAS).^{34 (link)} The
gel was incubated subsequently for 1 h in 12.5% (w/v) trichloroacetic
acid, 1 h in 1% (v/v) periodic acid/3% (v/v) acetic acid, 1 h in 15%
(v/v) acetic acid (replaced every 10 min), and 1 h at 4 °C in
the dark in Schiff’s reagent (Sigma-Aldrich, Zwijndrecht, The
Netherlands). Hereafter, the gel was washed two times for 5 min in
0.5% (w/v) sodium bisulfite and destained in 7% (v/v) acetic acid.
The isoelectric point (pI) of Chitinase Chi1 was
estimated by isoelectric focusing (IEF) using PhastGel IEF on a Pharmacia
LKB Phast System (Pharmacia Biotech, Uppsala, Sweden) with a broad
protein calibration kit (pH 3–10, GE Healthcare) as standard.
Proteins were stained with Coomassie blue R-2.