Protein Protocols Protein Protocols

274 Patton
plexes for colorimetric detection of electrophoretically separated proteins immobilized
on membranes (13–16). In 1978, a pink bathophenanthroline disulfonate-ferrous complex
was reported as a nonspecific protein stain for polyacrylamide gel electrophoresis
(20). The stain is rather insensitive and was later modified by substituting [ 59 Fe] into
the complex in order to detect proteins by autoradiography (21). Although increasing
sensitivity substantially, the hazards associated with working with radioactivity and
the burden of license application for an infrequently used radioisotope have precluded
routine utilization of bathophenanthroline disulfonate-[ 59 Fe] as a general protein stain.
Measuring light emission is intrinsically more sensitive than measuring light absorbance,
as the later is limited by the molar extinction coefficient of the colored complex
(28). Thus, luminescent protein detection systems utilizing chelates complexed to transition
metal ions such as europium, or ruthenium should offer greater sensitivity than
their colorimetric counterparts without the accompanying hazards associated with
radioactivity. The organic component of the complex absorbs light and transfers the
energy to the transition metal ion, which subsequently emits light at longer wavelength.
This is demonstrated by substituting europium into the bathophenanthroline–
disulfonate complex (17). This luminescent reagent has been commercialized as
SYPRO Rose protein blot stain (Molecular Probes, Eugene, OR). The bathophenanthroline
disulfonate-europium complex can detect as little as 8 ng of protein
immobilized on nitrocellulose or PVDF membranes. By comparison, the original
bathophenanthroline disulfonate–ferrous complex is capable of detecting 600 ng of
protein, while the modification employing [ 59 Fe] is capable of detecting 10–25 ng of
protein (20,21). The luminescent stain is readily removed by incubating blots in mildly
alkaline solution, is highly resistant to photobleaching and is compatible with popular
downstream biochemical characterization procedures including immunoblotting, lectin
blotting, and mass spectrometry (17). Disadvantages of the bathophenanthroline
disulfonate–europium stain are that the dye can only be adequately visualized using
302 nm UV-B epi-illumination and the dye exhibits intense 430 nm (blue) fluorescence
emission as well as the desired red emission maxima of 595 and 615 nm.
Subsequently, SYPRO Rose Plus protein blot stain, an improved europium-based
metal chelate stain roughly 10 times brighter than the original bathophenanthroline
disulfonate-europium stain, was introduced (25,26). The intense blue fluorescence from
uncomplexed ligand, observed in the original stain, was eliminated by employing a
thermodynamically more stable europium complex. Due to improved absorption
properties, the stain could now be readily visualized with UV-A UV-B or UV-C epiillumination.
Just as with the bathophenanthroline disulfonate–europium stain, SYPRO
Rose Plus stain is easily removed by increasing solution pH. The stain is fully compatible
with biotin–streptavidin and immunoblotting detection technologies that use a wide
variety of visualization strategies. Neither of the europium-based stains is compatible
with laser-based gel scanners as they lack visible excitation peaks. SYPRO Ruby dye is a
proprietary ruthenium-based metal chelate stain developed to address the limitations of
the SYPRO Rose and SYPRO Rose Plus dyes. SYPRO Ruby protein blot stain visualizes
electroblotted proteins on nitrocellulose and PVDF membranes with a detection
sensitivity of 0.25–1 ng of protein/mm 2 in slot-blotting applications. Approximately 2–8 ng
of protein can routinely be detected by electroblotting, which side-by-side comparisons
demonstrate is as sensitive as colloidal gold stain (22). While colloidal gold stain-