time of Amaranth was much less than the routine dyes and quick results were obtained; given in Table-1.
After staining the gel was destained by washing twice in destaining
solution (20% acetonitrile, 50 mM sodium acetate, and pH 4) for 30 minutes each time.
Followed by staining with ethidium bromide (1 ug/mL) for 30 minutes and destaining
with distilled water for 10 minutes.
The protein gels were fixed using destaining
solution immediately after electrophoresis and the protein bands were visualized under UV light (290 nm).
Staining and destaining
procedure was based on the pharmacia Phast Gel Developing Device.
Methylene blue is an alternative DNA stain that is noncarcinogenic but results in inferior staining and often requires more time for staining and destaining
(Herrin and Schmidt, 1988).
A final 0.025% staining/destaining solution was prepared by mixing 26.5 mL of stain stock with 184 mL destaining
solution (1:3:6 glacial acetic acid: methanol: distilled water) (12).
Samples including both the low and high range molecular markers were diluted at different ratios 1:3, 1:4, 1:5 and 1:6 (v/v) in loading buffer and run at a constant voltage of 150 for an hour after which the gel was stained in coomasie brilliant blue and the molecular weights of the protein subunits determined after destaining
for 2 hours, by a calibration graph obtained using the molecular weight markers.
After the electrophoresis, the gel slab was incubated in dark with 50 ppm Sigma's Stains-All in 50% ethanol for 24 h, rinsed three times by water, incubated in dark with water for 24 h, and exposed to light for 2 h for further destaining
. Finally, the treated agarose gel was viewed by an Alpha Innotech AlphaImager IS-2200 system, and the stained HA samples were analyzed by Alpha Innotech AlphaEase Spot Density software for electrophoretic mobility measurement.
The relative low cost of these dyes and their ready-made solutions, sensitivity in the 5-50 ng range and time-tested staining and destaining
protocols have been fundamental to their wide acceptance.
After staining, the gel was washed twice in distilled water to remove excess of the stain then the gel was covered with 200-250 ml of destaining
solution (45:45:10% Methanol: Acetic acid: Water).
The stain is inexpensive, is at least as sensitive as methylene blue, does not require destaining
, requires visualisation with only visible light, and is specific for DNA but not RNA.