Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 32)

(contd.)
Silver Stains
Three silver staining methods are recommended
for use with 2-D gels. Though they are based on
slightly different chemistries, they have similar
protein sensitivities.
The Bio-Rad silver stain kit, based on the method
of Merril et al. (1981), can be up to 100 times more
sensitive than Coomassie Blue R-250 dye staining
and allows visualization of heavily glycosylated
proteins in gels. Protein spots containing 10–100 ng
of protein can be easily seen. Proteins in gels are
fixed with alcohol and acetic acid, then oxidized
in a solution of potassium dichromate in dilute nitric
acid, washed with water, and treated with silver
nitrate solution. Silver ions bind to the oxidized
proteins and are subsequently reduced to metallic
silver by treatment with alkaline formaldehyde.
Color development is stopped with acetic acid
when the desired staining intensity has been
achieved. This method is not compatible with mass
spectroscopic analysis since the oxidative step
affects protein mass.
Silver Stain Plus stain from Bio-Rad, based on
the method developed by Gottlieb and Chavko
(1987), requires only one simultaneous staining and
development step. Proteins are fixed with a solution
containing methanol, acetic acid, and glycerol and
then washed extensively with water. The gels are
then soaked in a solution containing a silver-amine
complex bound to colloidal tungstosilicic acid.
Silver ions transfer from the tungstosilicic acid to the
proteins in the gel by means of an ion exchange or
electrophilic process. Formaldehyde in the alkaline
solution reduces the silver ions to metallic silver to
produce the images of protein spots. The reaction is
stopped with acetic acid when the desired intensity
has been achieved. Silver ions do not accumulate
within the gel, so background staining is light.
Since this method lacks an oxidizing step,
visualization of heavily glycosylated proteins and
lipoproteins can be less sensitive than with the
Merril stain.
Dodeca silver stain is based on the method
described by Sinha et al. (2001), in which protein-
bound silver ions are chemically reduced to form
visible metallic silver. This stain was developed for
use with the high-throughput Dodeca stainers and
can be used with mass spectrometry.
Stain-Free Technology
A special additive in Bio-Rad’s Criterion Stain Free ™ ,
Criterion ™ TGX Stain-Free ™ , and Mini-PROTEAN ®
TGX Stain-Free ™ gels covalently modifies tryptophan
residues when activated with UV light. This
enhances the proteins’ intrinsic fluorescence and
shifts the emission into the visible range (>400 nm),
allowing protein detection (with a stain-free
compatible imager, such as the Gel Doc
™ EZ or
ChemiDoc ™ MP systems) in a gel both before and
after transfer, as well as total protein detection on a
blot when using wet PVDF membranes.
This system is ideal for quick sample assessment
during purification procedures and as a precursor
to blotting and profiling workflows in which
Coomassie (Brilliant) Blue staining is ordinarily
used. The sensitivity of the Stain-Free system is
comparable to that of staining with Coomassie
Blue for proteins with a tryptophan content >1.5%;
sensitivity superior to Coomassie staining is possible
for proteins with a tryptophan content >3%.
Proteins that do not contain tryptophan residues
are not detected.
60 61
2-D Electrophoresis Guide Theory and Product Selection
Dodeca High-Throughput Stainers
Dodeca stainers are high-throughput gel staining
devices available in two sizes: the small size
accommodates up to 24 Criterion gels while the
large size can accommodate up to 12 large-format
gels. The stainers feature a shaking rack designed
to hold staining trays at an angle to allow air bubbles
to escape and ensure uniform gel staining to protect
gels from breaking. Use of the stainers ensures
high-quality, consistent results and eliminates
gel breakage from excess handling. They are
compatible with the following stains:
■ ■
Bio-Safe Coomassie (Brilliant) Blue G-250 stain
■ ■
Coomassie (Brilliant) Blue R-250 stain
■ ■
SYPRO Ruby protein gel stain
■ ■
Flamingo fluorescent protein gel stain
■ ■
Oriole fluorescent gel stain
■ ■
Dodeca silver stain kits
High-Throughput Dodeca Stainers
Detection of Proteins on Western Blots
Certain synthetic membranes bind proteins tightly
and can be used as supports for solid-phase
immunoassays, staining, or other analysis.
These membranes, known as western blots,
are useful for the identification of specific proteins
and protein modifications.
2-D electrophoresis can be combined with
western blotting for monitoring the posttranslational
modification of trace proteins in complex mixtures and
evaluating the specificity of antibodies and antisera.
Numerous techniques are available for the transfer of
proteins to membranes and for the probing of western
blots with antibodies, stains, and other reagents.
These techniques are described in more detail in
the Protein Blotting Guide (bulletin 2895).
Chapter 5: Detection