Protein Protocols Protein Protocols

770 Packer et al.
9. The postlabeling procedure is used for proteins that have already been separated by gel
electrophoresis and immobilized on blots. It is important to use PBS (buffer E) instead of
TBS (buffer A) in the initial stages as Tris inhibits the DIG labeling process.
10. Nitrocellulose and PVDF membranes can be used. Nylon membranes result in high background.
11. For mucins and other heavily glycosylated proteins it may be necessary to fix the bands to
the membrane before labeling by washing in 1% KOH for 5 min. If this procedure is done,
increase the initial washes to 3 × 10 min in buffer B.
12. Postlabeling results in sharper bands than prelabeling but has a lower sensitivity and
requires more DIG (5 µL per gel).
4.3. DIG-Labeled Lectin Staining
13. The binding of some lectins will be increased by desialylation while the binding of others
will be decreased.
14. Suggested dilutions of the DIG labeled lectins: SNA—1:1000, MAA—1:500, DSA—
1:1000, PNA—1:100.
15. The divalent ions are necessary for optimal lectin reactivity. The stock solution should be
diluted to give a final concentration of 1 mM of each ion.
4.4. Pro-Q Emerald 300 Dye Staining
16. The Pro-Q Emerald 300 stain has an excitation maximum at approx 280 nm and an emission
maximum near 530 nm. Stained glycoproteins can be visualized using a 300 nm UV
transilluminator. The use of a photographic camera or charge coupled device (CCD) camera
and the appropriate filters is essential to obtain the greatest sensitivity.
17. It is important to clean the surface of the transilluminator after each use with deionized
water and a soft cloth (such as cheesecloth). Otherwise, fluorescent dyes can accumulate
on the glass surface and cause a high background fluorescence.
18. Use a 300 nm transilluminator with six 15W watt bulbs. Excitation with different light
sources may not give the same sensitivity.
19. Using a Polaroid ® camera and Polaroid 667 black-and-white print film, the highest sensitivity
is achieved with a 490 nm longpass filter, such as the SYPRO protein gel stain
photographic filter (S-6656), available from Molecular Probes. Gels are typically photographed
using an f-stop of 4.5 for 2–4 s, using multiple 1-s exposures.
20. Using a CCD camera, images are best obtained by digitizing at about 1024 × 1024 pixels
resolution with 12–, 14– or 16– bit gray scale levels per pixel. A 520 nm long pass filter is
suitable for visualizing the stain. A CCD camera-based image analysis system can gather
quantitative information that will allow comparison of fluorescence intensities between
different bands or spots. Using such a system, the Pro-Q Emerald stain has a linear dynamic
range over three orders of magnitude. The polyester backing on some premade gels is
highly fluorescent. For maximum sensitivity using a UV transilluminator, these gels
should be placed polyacrylamide side down and an emission filter used to screen out the
blue fluorescence of the plastic.
21. The green-fluorescent Pro-Q Emerald 300 staining should be viewed and documented
before staining total proteins with SYPRO Ruby protein gel stain. Pro-Q Emerald dye
signal will fade somewhat after SYPRO Ruby dye staining.
4.5. HPAEC Analysis of Monosaccharide Composition
22. Data on the composition of both the acidic sialic acids and the hexoses and amino sugars
can be obtained sequentially from a single spot. After the 4 M TFA hydrolysis,