Protein Protocols Protein Protocols

776 Møller and Poulsen
gel can be photographed sequentially on a light box during the development step for identification
of proteoglycans. As a control, a gel can be stained for proteins with ordinary
silver (Subheading 3.1., steps 3–9), in which proteoglycans will not stain.
2. The methods are optimized for the PhastSystem, which uses small, supported gels that are
stained in an automatic development chamber. If larger gels are stained in staining trays at
room temperature, longer incubation times are needed. For unsupported 1-mm thick gels,
good results are obtained by increasing the washing with washing solution I in steps 1
and 3 (Subheading 3.1.) to a total of 2.5 and 1.5 h, respectively, with four changes of the
solution, and furthermore doubling the incubation times in all other steps.
3. Handle gels with gloves or forceps, as fingerprints stain. The staining methods are highly
sensitive, and it is essential that the equipment (development chamber/staining trays) is
scrupulously clean and that high-quality water is used. Use a separate tray for the staining
solution. Gels should be agitated during all staining and washing procedures.
4. This rather extensive washing procedure is necessary to remove the SDS from the gel,
which otherwise precipitates alcian blue, resulting in excessively high background. If gels
are run in native PAGE, one short washing step is sufficient.
5. Alcian blue is irreversibly fixed in the gel by the subsequent silver staining and results in
a greenish-black background if the stain is not washed out by dilute acetic acid. Only a
weak bluish nuance in the background should remain.
6. Glutaraldehyde is injurious to health. If the procedure is not carried out in a closed chamber,
a fume cupboard should be used.
7. This step is important for washing out excess silver ions without losing silver bound to
Alcian blue/proteoglycan for autocatalytic reduction in the development step. Too little
washing leads to formation of metallic silver in the background, whereas too intense washing
leads to decreased sensitivity.
8. The image is stable, but over time, increased background staining will develop, especially
from light exposure.
9. In this staining procedure, Acetic acid/ethanol solutions are not always efficient for fixation
of the proteins in the gel, whereas Trichloroacetic acid works well.
Acknowledgments
This work was supported by the Danish Rheumatism Association and the Danish
Medical Research Council.
References
1. Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) A highly sensitive silver stain for
detecting proteins and peptides in polyacrylamide gels. Analyt. Biochem. 98, 231–237.
2. Heinegård, D. and Sommarin, Y. (1987) Isolation and characterization of proteoglycans,
in Methods in Enzymology, Vol. 144: Structural and Contractile Proteins (Cunningham,
L. W., ed.), Academic, Press, New York, pp. 319–372.
3. Zacharius, R. M., Zell, T. E., Morrison, J. H., and Woodlock, J. J. (1969) Glycoprotein
staining following electrophoresis on acrylamide gels. Analyt. Biochem. 30, 148–152.
4. Wardi, A. H. and Michos, G. A. (1972) Alcian blue staining of glycoproteins in acrylamide
disc electrophoresis. Analyt. Biochem. 49, 607–609.
5. Eckhardt, A. E., Hayes, C. E., and Goldstein, I. J. (1976) A sensitive fluorescent method
for the detection of glycoproteins in polyacrylamide gels. Analyt. Biochem. 73, 192–197.
6. Van-Seuningen, I. and Davril, M. (1992) A rapid periodic acid-Schiff staining procedure
for the detection of glycoproteins using the PhastSystem. Electrophoresis 13, 97–99.