Protein Protocols Protein Protocols

282 Patton
EDTA, pH 9.6 or using SYPRO Rose Plus destain solution (component C). All the
metal chelate stains mentioned above may be destained using the following protocol:
1. Incubate the membrane in appropriate destain reagent for 15 min with continuous gentle
agitation.
2. Rinse twice for 1 min each in deionized water.
4. Notes
4.1. Colorimetric Detection of Proteins on Nitrocellulose, PVDF,
or Nylon Membranes
1. Although enhancement of the Ferrozine–ferrous stain with ferrocyanide–ferric stain substantially
increases detection sensitivity, the double stain is also more difficult to elute than
the Ferrozine–ferrous stain alone (see Subheading 3.7. for methods of stain reversal).
2. The ferrocyanide-ferric stain may form a precipitate after long–term storage. The precipitate
is easily resuspended by vigorous shaking or sonication.
3. If a dried nitrocellulose membrane is incubated in the ferrocyanide–ferric stain, a patchy
background may result that is difficult to destain. Dry blots should be rehydrated briefly in
deionized water prior to incubation in the ferrocyanide–ferric stain.
4. The number of washes necessary to remove background staining may vary slightly. The
ferrocyanide–ferric stain may initially remain bound to the nitrocellulose membrane but
the membrane background will become white with sufficient washing.
5. Membranes may be imaged transmissively or reflectively. Transmissive imaging is preferred
over conventional reflective imaging as it improves signal detection while maintaining
a white background. Furthermore, in some cases the protein sample penetrates
through the membrane support to the reverse side (particularly with vacuum slot blotters).
The metal chelate complexes stain proteins present throughout the thickness of the membrane
and on both surfaces but reflective scanning only detects signal on the front surface,
leading to a poorer linear dynamic range of quantitation. This problem is alleviated with
transmissive scanning. Typically, a 45 W white light box is used in conjuction with a
450 ± 70 nm bandpass filter to enhance image contrast when imaging Ferrozine–ferrous
or Pyrogallol Red-molybdenum stains. For blue stains such as Ferrozine-ferrous followed
by the ferrocyanide-ferric stain, a 600 ± 70 nm bandpass filter may enhance the image
contrast. Because the membrane support blocks a substantial amount of transmitted light, care
must be taken to completely mask the sample with black cardboard for transmissive imaging.
4.2. Luminescent Detection of Electroblotted Proteins Using
Bathophenanthroline Disulfonate-Europium (SYPRO Rose Stain)
6. Since formate ions may chelate europium ions, the washes with deionized water are crucial
for complete removal of the formate buffer. Otherwise, the staining solution may be
inactivated. Care should be taken to aspirate off all the solution between washes.
7. Removal of unbound bathophenathroline-europium stain can not be visually monitored as
with the colorimetric stains. We have found that washing 4× for 1 min is effective. However,
the number and duration of washes may vary from case to case (depending on the
size of the membrane and the volume of each wash). Therefore, optimal washing should
be determined empirically for each application.
8. After staining, wet membranes should not be touched since residue found on latex laboratory
gloves may destroy the stain. Once dry, membranes can be handled freely. Since