Katheryn Resing
III. Rapid Reverse Stain with Imidazole/Zn++
From: Fernandez-Patron C., Castellanos-Serra, L and Rodriguez, P. (1992)
Biotechniques 12: 564-573 Procedure:
Protocol written by David F. Smith, Pharmacology, UNMC
To Stain:
1. Remove slab gel from unit and rinse briefly with dH2O
2. Equilibrate on shaker for 5 min. in 50 -100 milliliters of 0.2 M
imidazole (13.6 g/liter).
3. Stain for approximately one minute in 50-100 milliliters of 0.3 M
ZnCl2 (41 g/liter *); observe on a black background.
4. Wash with dH2O
* To dissolve ZnCl2, carefully adjust pH to 5.0; Zinc sulfate or
acetate will work equally well.
Options following staining:
1. If necessary, destain with 2% citric acid for 10 min.
2. Individual gel bands or spots can be excised, then destained; (For
tryptic/ Lys-c digest to follow: destain gel piece with 1 ml 2% citric
acid for 10 minutes at room temperature on rocker.). Equilibrate in
appropriate buffer for further processing following destaining.
3. Destained gel can be equilibrated in appropriate buffer for
electroblotting to PVDF.
4. Destained gel can be stained by normal Coomassie procedures.
Advantages:
This procedure is particularly suited of identifying gel spots or
bands that will be electroeluted for microsequence analysis or that will
be
subsequently transferred to PVDF for Western blots. The staining is more
sensitive than normal Coomassie staining, actually approaching the
sensitivity of silver stain and extremely rapid (staining and destaining
can take as little as 15 minutes.
Zn++ staining does not displace SDS from the proteins or otherwise
fix the proteins in the gel matrix, so electroelution or electroblotting
should yield maximal recovery of protein. In one case, DFS destained a
gel 3 days after Zn++ staining and got excellent Western blot results
following
transfer to PVDF.
Stained gels can be scanned (DFS uses a Molecular Dynamics
Personal Densitometer) to digitize the gel image. The image can then be
reversed using image editing software to give an excellent, high-contrast
gel image with black bands on a white background.
For in gel digests, destain with 2% citric acid for 10 minutes, then
procede with in-gel digest protocol (include the first destain step because
it removes the SDS).