Protein Protocols Protein Protocols

764 Packer et al.
c. Solution 3: 75 mg/mL of 4-nitrobluetetrazolium chloride dissolved in 70% (v/v)
dimethylformamide.
d. Solution 4: 50 mg/mL of 5-bromo-4-chloro-3-indolyl-phosphate dissolved in dimethylformamide.
Store these solutions (3 and 4) in the dark.
2.4 Pro-Q Emerald 300 Dye Staining
1. Pro-Q Emerald 300 glycoprotein gel stain kit (Molecular Probes, OR, USA) containing:
a. Pro-Q Emerald 300 reagent, 50× concentrate in dimethyl formamide (DMF) (component
A), 5 mL.
b. Pro-Q Emerald 300 dilution buffer (component B), 250 mL.
c. Periodic acid (component C), 2.5 g.
d. SYPRO Ruby protein gel stain (component D), 500 µL.
e. SDS, component E), 500 µL of a 10% solution.
f. CandyCane glycoprotein molecular weight standards (component F), 40 µL, sufficient
volume for approx 20 gel lanes. Each protein is present at approx 0.5 mg/mL. The
standards contain a mixture of glycosylated and nonglycosylated proteins, which, when
separated by electrophoresis, provide alternating positive and negative controls.
2. Fix solution: Prepare a solution of 50% methanol and 50% dH 2O. One 8 × 10 gel will
require approx 100 mL of fix solution.
3. Wash solution. Prepare a solution of 3% glacial acetic acid in dH 2O. One 8 × 10 gel will
require about approx 250 mL of wash solution.
4. Oxidizing solution. Add 250 mL of 3% acetic acid to the bottle containing the periodic
acid (component C) and mix until completely dissolved.
5. CandyCane molecular weight standards diluted in sample buffer. For a standard lane on a
8 cm × 10 cm gel, dilute 0.5 µL of the standards with 7.5 µL of sample buffer and vortexmix.
This will result in approx 250 ng of each protein per lane, a sufficient amount for
detection of the glycoproteins by the Pro-Q Emerald 300 stain. For large 16 cm × 18 cm
gels, double the amount of standard and buffer used.
2.5. HPAEC Analysis of Monosaccharide Composition
1. Methanol.
2. 0.1 M TFA.
3. 2 M TFA.
4. Standard sugars:
a. 0.1 mmole/mL of lactobionic acid.
b. 0.1 mmole/mL of 2-deoxyglucose.
c. 0.1 mmole/mL of N-acetyl neuraminic acid and N-glycolyl neuraminic acid.
d. 0.1 mmole/mL of mixture of fucose, 2-deoxyglucose, galactosamine, glucosamine,
galactose, glucose and mannose.
Sugars should be dried thoroughly over phosphorus pentoxide in a desiccator before
weighing.
5. Metal-free HPLC System with DIONEX CarboPac PA1 PA10 column, 4 mm × 25 cm and
pulsed amperometric detector (HPAEC-PAD).
3. Methods
3.1. Periodic Acid–Schiff Staining
This method is essentially as described in ref. 3 for glycoproteins transferred to
nitrocellulose membranes. A modification of the method for PVDF membranes is
described in ref. 4.