Protein Protocols Protein Protocols

262 Choi, Hong, and Yoo
Fig. 2. Mechanism of protein–dye interaction in acidic solution.
Fig. 3. Structure of NN.
4. The rate of destaining speeds up with increasing methanol content; however, at high
methanol content (>55%), gels are opaqued and shrunken. Addtionally, increasing the
temperature of destaining solution is a great help in removing background (at 60–70°C, in
5 min), although sensitivity is a little reduced.
5. Destaining can be completed in 30 min in 7.5% polyacrylamide gels, but destaining time
should be increased for 10 and 12.5% gels (50–60 min).
6. NN has several functional groups, such as hydroxyl, diazoic, carboxyl, and sulfonate
groups (see Fig. 3). At acidic pH, NN probably forms electrostatic bonds with protonated
amino groups, which are stabilized by hydrogen bonds and Van der Waals forces, as
does CB (1).
7. For maximal staining effect, the dye solution should be freshly prepared. The preparation
of staining solution requires stirring and warming at 50–60°C since NN is poorly soluble.
8. Bands stained with NN are indefinitely stable when gels are stored in a refrigerator
wrapped up in polyethylene films or dried on Whatmann No. 1 filter paper.
9. Throughout the staining/destaining processes, it is necessary to agitate the gel container
using a shaker.
Acknowledgments
This work has been supported by a grant from KOSEF (981-0704-033-2) to J. K.
Choi in 1988. This work has been supported by a Korean Research Foundation Grant
(KRTF-2000-041-00301) to J. K. Choi in 2000.
References
1. Fazekas de St. Groth, S., Webster, R. G., and Datyner, A. (1963) Two new staining procedures
for quantitative estimation of proteins on electrophoretic strips. Biochim. Biophys.
Acta 71, 377–391.