Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 31)

(contd.)
Coomassie Stains
Coomassie R-250 staining solution is prepared for
a traditional staining procedure in which gels are
stained in a methanol-water-acetic acid solution of
Coomassie R-250 dye. It requires ~40 ng protein
per spot for detection, though absolute sensitivity
and staining linearity depend on the proteins
being stained.
Bio-Safe Coomassie stain is a ready-to-use, single-
reagent protein stain made with Coomassie (Brilliant)
Blue G-250. It offers sensitivity similar to colloidal
Coomassie stains (down to 8 ng) and a rapid
staining protocol. No additional reagents besides
water are required.
Fluorescent Stains
Flamingo fluorescent gel stain is prepared from
a dye that binds denatured protein. Normally
non-fluorescent in solution, it becomes strongly
fluorescent when bound to protein. There is,
therefore, no need for destaining, since unbound
dye in the gel is only minimally fluorescent.
A prolonged fixing step is necessary to wash
buffers and SDS out of the gel prior to staining,
as these substances can prevent dye binding.
Flamingo fluorescent gel stain is the most sensitive
of the listed fluorescent stains, with sensitivity to
0.25–0.5 ng, and it can be linear over three orders of
magnitude. The simple two-step staining procedure
can be completed in as little as five hours.
With a primary fluorescence excitation maximum at
512 nm and a considerably weaker excitation peak
at 271 nm, Flamingo fluorescent gel stain gives
the most sensitive results when imaged with laser
fluorescence scanning instruments equipped with
green or blue laser light sources. UV transilluminator-
based systems may also be used, but extended
exposure times may be required and sensitivity will
not be as high.
Oriole fluorescent gel stain is sensitive and, of the
stains listed, it is the easiest and most rapid to use.
The one-step staining process does not require
fixation or destaining, allowing protein samples
to be accurately visualized and quantitated in less
than two hours. Since SDS is required for optimal
staining, prior fixing or washing of the gel can impair
staining sensitivity.
The dye in Oriole stain is excited only weakly by
wavelengths longer than 400 nm and can, therefore,
only be imaged using UV-based imaging systems.
Oriole’s limit of detection is 1 ng or less in a typical
protein spot.
SYPRO Ruby was one of the original fluorescent
protein gel stains, and it has a combination of high
sensitivity and wide dynamic range that cannot be
achieved with traditional Coomassie blue or silver
stains. SYPRO Ruby has two prominent absorbance
peaks, one at ~270 nm in the UV range and the
other at ~460 nm in the visible range. This allows
imaging with both UV transilluminator and laser-
scanning systems. Detection sensitivity in SYPRO
Ruby–stained gels can be as low as 1 ng. SYPRO
Ruby stains most classes of proteins with little
protein-to-protein variability.
The principle advantage of SYPRO Ruby is its
versatility with respect to imaging requirements.
It is, however, time-consuming to use and does
not produce the high-quality mass spectrometric
data generated with other fluorescent stains
(Berkelman et al. 2009).
Protein Stains
Bio-Rad total protein stain selection guide.
Detection Sensitivity MS
Total Protein Stain Method (Lower Limit) Time Comments Compatible?
Coomassie Stains Visible
Coomassie (Brilliant) Blue 36–47 ng 2.5 hr Simple and consistent; requires destaining Yes
R-250 with methanol
Bio-Safe ™ Coomassie 8–28 ng 1–2.5 hr Nonhazardous staining in aqueous Yes
G-250 solution; premixed
Silver Stains Visible
Silver stain 0.6–1.2 ng 2 hr Stains glycoproteins, lipoproteins, No
(Merril et al. 1981) lipopolysaccharides, nucleic acids
Silver Stain Plus ™ kit 0.6–1.2 ng 1.5 hr Simple, robust protocol Limited
(Gottlieb and Chavko 1987)
Dodeca ™ silver stain kit 0.25–0.5 ng 3 hr Simple, robust protocol; ideal for use Yes
with Dodeca stainers (Sinha et al. 2001)
Fluorescent Stains Fluorescence
Oriole fluorescent 0.5–1 ng 1.5 hr Rapid protocol requires no fixing or Yes
gel stain destaining; requires UV excitation
Flamingo fluorescent 0.25–0.5 ng 5 hr Simple protocol requires no destaining; Yes
gel stain high sensitivity, broad dynamic range;
excellent for laser-based scanners
SYPRO Ruby protein 1–10 ng Overnight Simple, robust protocol; Yes
gel stain broad dynamic range
Negative Stains Visible
Zinc stain 6–12 ng 15 min High-contrast results; simple, fast, and Yes
reversible; compatible with elution or blotting
as well (Fernandez-Patron et al. 1992)
Copper stain 6–12 ng 10 min Single reagent; simple, fast protocol and Yes
reversible stain; compatible with elution or
blotting as well (Lee et al. 1987)
Stain-Free Technology Stain-Free 8–28 ng 5 min No separate staining steps Yes, but
fluorescence tryptophan
residues are
modified
Coomassie Brilliant Blue R-250 Silver Stain Kit Silver Stain Plus Kit
Zinc Stain and
Destain Kit
Flamingo Fluorescent
Gel Stain
Oriole Fluorescent
Gel Stain
Copper Stain
58 59
2-D Electrophoresis Guide Theory and Product Selection Chapter 5: Detection