Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 30)

56 57
2-D Electrophoresis Guide Theory and Product Selection Chapter 5: Detection
■ ■
For quantitative comparisons, use stains with broad
linear ranges of quantitation (for example, Flamingo
™ ,
Oriole ™ , and SYPRO Ruby)
■ ■
Since no stain is capable of staining all proteins,
consider staining replicate gels with two or more
different stains. Coomassie (Brilliant) Blue appears
to stain the broadest spectrum of proteins.
Therefore, it is instructive, especially with 2-D gels,
to stain a Coomassie Blue–stained gel with silver,
or to stain a fluorescently stained gel with colloidal
Coomassie Blue or silver. Often, this double staining
procedure reveals a few differences between the
protein patterns. It is possible to stain gels first with
Coomassie Blue or a fluorescent stain, then again
with silver
The sensitivity achievable in staining is determined by:
■ ■
The amount of stain that binds to the proteins
■ ■
The intensity of the coloration
■ ■
The difference in color intensity between stained
proteins and the residual background in the body
of the gel (the signal-to-noise ratio); unbound stain
molecules can be washed out of the gels without
removing much stain from the proteins
No stain interacts with all proteins in a gel in precise
proportion to their mass, and all stains interact
differently with different proteins (Carroll et al. 2000).
The only observation that seems to apply for most
stains is that they interact best with proteins with
a high basic amino acid content.
Coomassie Stains
Coomassie (Brilliant) Blue is the most common
stain for protein detection in polyacrylamide gels.
Coomassie R-250 and G-250 are fabric dyes that have
been adapted to stain proteins in gels. The “R” and “G”
designations indicate red and green hues, respectively.
These stains generate visible protein patterns that can
be analyzed using densitometric methods.
Detection of Proteins in Gels
In 2-D electrophoresis, proteins in gels are most
commonly visualized using total protein stains.
Selection of the most appropriate stain involves
consideration of the stain characteristics, limitations
with regard to the sensitivity of detection and the types
of proteins it stains best, downstream applications,
and the type of imaging equipment available
(see Chapter 6). For use in proteomics applications,
stains should be compatible with high-throughput
protocols and downstream analysis, including
digestion and mass spectrometry (Patton 2000).
It is also possible to label protein samples after sample
preparation and prior to IEF with fluorescent dyes such
as the CyDye DIGE fluors (Westermeier and Scheibe
2008). At the time of writing, three dyes with spectrally
different excitation and emission wavelengths were
available, allowing labeling of up to three different
samples and their separation in a single 2-D gel.
The dyes are matched for size and charge to obtain
migration of differently labeled identical proteins to the
same spot positions. The labeled samples are mixed
together before they are applied on the gel of the first
dimension. After separation, the gels are scanned with
fluorescence imagers at the different wavelengths.
The following are general tips for staining 2-D gels:
■ ■
2-D gels are clearer, sharper, and more reproducible
when less protein is loaded. When sample
preparation and IEF conditions are not optimized,
it is often beneficial to load relatively little protein and
to use a relatively sensitive staining technique
■ ■
To identify low-abundance proteins, apply a high
protein load and use a high-sensitivity stain
(for example, silver or a fluorescent stain)
(Corthals et al. 2000)
■ ■
To obtain enough protein for mass spectrometry,
apply a high protein load and use a compatible
staining procedure
Silver Stains
Silver stains offer high sensitivity but with a low
linear dynamic range (Merril et al. 1981). Often, these
protocols are time-consuming and complex. Silver
staining protocols have multiple steps with critical
timing; for this reason, they can be insufficiently
reproducible for quantitative analysis. In addition,
their compatibility with mass spectrometric protein
identification techniques is lower than Coomassie
stains and fluorescent dyes. There are many different
silver staining techniques with differing chemistries
and sensitivities.
Fluorescent Stains
Fluorescent stains fulfill almost all the requirements for
an ideal protein stain by offering high sensitivity, a wide
linear dynamic range (up to four orders of magnitude),
a simple and robust protocol, and compatibility with
mass spectrometry. These sensitive stains generate
little background and are easy to use.
Because fluorescent stains require specialized
instrumentation for imaging, the choice of stain
may be dictated by the instrumentation available.
Fluorescent dyes absorb light at one wavelength
and re-emit the light at another longer wavelength.
Imaging instruments differ in both the type of light
delivered for absorbance and the capabilities for
detecting the emitted light. The simplest and least
expensive systems use UV transillumination and a
camera for image capture; however, not all fluorescent
stains are optimally excited by UV light. Other imaging
systems use laser light to scan the gel. Laser light
is monochromatic, and the laser must be selected
according to the absorbance properties of the dye.
Not all fluorescent gel stains absorb visible light at
wavelengths supplied by imaging lasers.
Fluorescent stains can be at least as sensitive as
silver stains and are, therefore, subject to some of
the same potential problems stemming from high
sensitivity. Clean technique is essential, as any dust or
dirt transferred to the surface of the gel may appear
in the fluorescence image as smudges or speckles.
Contaminant proteins such as keratin will also appear
in the gel image if care is not taken to minimize such
contamination.
All fluorescent reagents are subject to photobleaching
to varying degrees. The fluorescent stains discussed in
the Protein Stains sidebar are reasonably photostable
and do not degrade noticeably through routine
exposure to room light during a staining procedure.
However, avoid exposure of the gel or staining solution
to intense light and cover the staining tray with an
opaque lid or foil.
Negative Stains
These rapid stains require only ~15 min for high-
sensitivity staining and generate protein bands that
appear as clear areas in a white background. Zinc and
copper stains do not require gel fixation and proteins
are thus not altered or denatured. Negative stains
can be used as a quality check before transferring
to a western blot or analysis by mass spectrometry,
though they are not the best choice when quantitative
information is desired.
Stain-Free ™ Technology
This proprietary Bio-Rad technology allows protein
detection in a gel both before and after transfer,
as well as total protein detection on a blot when
using wet PVDF membranes, without the need for
application of a stain (see sidebar).