Protein Protocols Protein Protocols

896 Turnbull
13. Enzyme buffer (0.2 M Na acetate, pH 4.5). Make 0.2 M sodium acetate (27.2 g/L of
sodium acetate trihydrate) and 0.2 M acetic acid (11.6 mL/L) and mix in a ratio of 45 mL
to 55 mL, respectively.
14. Enzyme stock solutions (typically at concentrations of 500 mU/mL, where 1 U = 1 µmol
substrate hydrolyzed per minute). Available from Glyko, Novato, CA.
15. Vortex tube mixer.
16. Microcentrifuge.
17. Acrylamide stock solution (T50%–C5%). Caution: Acrylamide is neurotoxic. Wear
gloves (and a face mask when handling powdered forms). It is convenient to use premixed
bis-acrylamide such as Sigma A-2917. Add 43 mL of distilled water to the 100-mL bottle
containing the premixed chemicals and dissolve using a small stirrer bar (approx 2 h).
The final volume should be approx 80 mL. Store the stock solution at 4°C. Note that it is
usually necessary to warm gently to redissolve the acrylamide after storage.
18. Resolving gel buffer stock solutions: 2 M Tris-HCl, pH 8.8 (242.2 g/L of Tris base; adjust
pH to 8.8 with HCl).
19. Stacking gel buffer stock solution: 1 M Tris-HCl, pH 6.8 (121.1 g/L of Tris base; adjust
pH to 6.8 with HCl).
20. Electrophoresis buffer (25 mM Tris, 192 mM glycine, pH 8.3): 3 g/L of Tris base, 14.4 g/L
of glycine; adjust pH to 8.3 if necessary with HCl.
21. 10% Ammonium persulfate in water (made fresh or stored at –20°C in aliquots).
22. N,N,N',N'-Tetramethylethylenediamine (TEMED).
23. Vertical slab gel electrophoresis system (minigel or standard size).
24. D.C. Power supply unit (to supply up to 500–1000 V and 200 mA).
25. UV transilluminator (312 nm maximum emission wavelength).
26. Glass UV bandpass filter larger than gel size (type UG-11, or M-UG2).
27. Charge coupled device (CCD) imaging camera fitted with a 450-nm (blue) bandpass filter.
3. Methods
3.1. Tagging Saccharides with a Fluorophore
HS (and heparin) saccharides can be endlabeled by reaction of their reducing aldehyde
functional group with a primary amino group of a fluorophore (reductive
amination). For sulfated saccharides anthranilic acid (2-AA; 11) has been found to be
effective for the IGS methodology. 2-AA conjugates display an excitation maxima in
the range 300–320 nm, which is ideal for visualization with a commonly available
312-nm UV source (e.g., transilluminators used for visualizing ethidium bromide
stained DNA). Emission maxima are typically in the range 410–420 nm (bright violet
fluorescence). Recently it has been found that approx 10-fold more sensitive detection
is possible using an alternative fluorophore ANDSA (16). ANDSA has an excitation
maxima of 350 nm and emission maxima of 450 nm. Both approaches described in
Subheadings 3.1.1. and 3.1.2. allow rapid labeling and purification of tagged saccharide
from free tagging reagent, and give quantitative recoveries and products free of salts that
might interfere with subsequent enzymic conditions. For saccharides in the size range hexato
dodecasaccharides, approx 2–3 nmol (approx 2–10 µg) of purified starting material is the
minimum required using the 2-AA label and approx 5- to 10-fold less for ANDSA labeling.
3.1.1. Labeling Saccharides with 2-AA
1. Dry down the purified saccharide (typically 2–10 nmol) in a microcentrifuge tube by centrifugal
evaporation.