White Page Gels

by Harald Zähringer, Labtimes 05/2012

The pioneer of histology staining, Paul Ehrlich, used methylene blue as early as 1885 to stain the substantia nigra in brain tissue.

Methylene blue has been used to stain cellular components for more than 100 years. However, it may also be applied to stain nucleic acids in PAGE-gels.

Methylene blue can be used for the staining of nucleic acids in polyacrylamide gels as a cheap and non-toxic alternative to the frequently applied mutagenic dye, ethidium bromide. The major reason why people shy away from methylene blue staining of DNA in PAGE-gels becomes quite obvious, if you look at the PAGE-gel a few hours after the methylene blue staining: the methylene blue bands have simply vanished.

The binding of methylene blue to DNA seems to be pretty weak; hence, the blue bands disappear within a few hours. They may be preserved when the gel is dried quickly in a special gel dryer, however, not every lab has a gel dryer available. But do not despair; the dryer is not necessarily required. By applying a simple trick, developed by Maria Soto and David Draper from the Johns Hopkins University, the methylene blue dye can be fixed in air-dried gels (Anal. Biochem., 2012, 421, 345-6).

Repeated washing

To this end, the gel is stained as usual in a solution of 0.03% methylene blue and 300 mM NaAc (pH 5.2) for about 15 minutes or until it turns blue. It is subsequently de-stained in water, until it appears transparent, with only the blue bands of the methylene blue stained DNA visible. Now for Soto’s trick: the gel is soaked in 95% ethanol for 25 minutes. During this period the ethanol is changed three times. An 8 x 9 mini-gel, e.g., is soaked in 8 ml ethanol for 10 minutes before the ethanol is discarded and replaced with another 8 ml of fresh ethanol. The ethanol is changed again after a further 10 minutes and rests in the tray for a final five minutes. The gel turns white upon this treatment and shrinks to about 6 x 6.5 centimetres.

The gel is wrapped into plastic foil and manually dried for about 30 minutes. During this time, the wrapped gel is straightened and turned until it becomes rubber-like in texture. Finally, the gel is unwrapped, charged with a light weight to prevent curving and air-dried overnight. The dried gel can be stored without losing the blue bands. And there is yet another advantage to this method: the white colour and the shrinking of the gel, which doesn’t affect the gel’s length-to-width ratio, significantly increase the visibility of the blue bands.

Methylene blue stained PAGE-gels before (l.) and after fixation of the bands. Gels were loaded with different amounts of short RNA-fragments. The bands are better visible on the shrinked white gel (r.) than on the original gel (l.).

Last Changed: 10.11.2012