Protein Protocols Protein Protocols

Transition Metal Chelate Stains 283
water is known to quench europium luminescence, drying the membrane also serves to
enhance the signal (about twofold).
9. Images are best obtained using a cooled CCD-camera by digitizing at about 1024 × 1024
picture elements (pixels) resolution with 12- or 16-bit gray scale levels assigned per pixel.
To visualize the stain, the front face of membranes should be illuminated with the UV
source: a hand-held, UV-B (302 nm) light, a UV light box placed on its side, or a top
illuminating system such as the Bio-Rad Fluor-S imager. Direct transillumination through
the blotting membrane yields unsatisfactory results. SYPRO Rose protein blot stain is
best visualized using a 490-nm longpass filter such as a SYPRO protein gel photographic
filter (Molecular Probes, Inc., cat. no. S-6656) or a 600-nm bandpass filter.
4.3. Luminescent Detection of Electroblotted Proteins
Using SYPRO Rose Plus Protein Blot Stain
10. Removal of unbound bathophenathroline–europium stain cannot 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.
11. Proteins stained with SYPRO Rose Plus protein blot stain are readily visualized by eye
using epi-illumination with a UV light source. Illuminate the front face of the membranes
using a hand-held, UV-B (302 nm) light source, a UV light box placed on its side, or a top
illuminating system such as the Bio-Rad Fluor-S imager. For greatest sensitivity and
accurate quantitation, photograph the blot using Polaroid photography with a 490-nm
longpass filter such as the SYPRO protein gel photographic filter (Molecular Probes, Inc.,
cat. no. S-6656) or use a computerized CCD camera-based image analysis system equipped
with a 600-nm bandpass filter. Direct transillumination through the blotting membrane
yields unsatisfactory results.
4.4. Luminescent Detection of Electroblotted Proteins
Using SYPRO Ruby Protein Blot Stain
12. Proteins stained with SYPRO Ruby protein blot stain are readily visualized by eye using
epi-illumination with UV light source. Illuminate the front face of the membranes using a
hand-held, UV-B (302 nm) light source, a UV light box placed on its side, or a top illuminating
system. Alternatively, use direct transillumination through the blotting membrane.
For greater sensitivity and accurate quantitation, photograph the blot using Polaroid ® photography
or a computerized CCD camera-based image analysis system equipped with a
490-nm longpass filter such as the SYPRO protein gel photographic filter (Molecular
Probes, Inc., cat no. S-6656) or a 600-nm bandpass filter. Laser-based imaging systems
can also be used.
13. Images are best obtained using a cooled CCD-camera by digitizing at about 1024 × 1024
picture elements (pixels) resolution with 12- or 16-bit gray scale levels assigned per pixel.
To visualize the stain, the front face of membranes should be illuminated with the UV
source: a hand-held, UV-B (302 nm) light, a UV light box placed on its side, or a top
illuminating system such as the Bio-Rad Fluor-S imager. Satisfactory results are obtained
from direct transillumination through the blotting membrane as well. SYPRO Ruby protein
blot stain is best visualized using a 490-nm longpass filter such as a SYPRO protein
gel photographic filter (Molecular Probes, Inc., cat. no. S-6656) or a 600 nm bandpass