Biomedical Engineering Reference
In-Depth Information
ultrapure water. Wash reagents are usually prepared using 20% v/v of methanol or ethanol
in water.
2. Sensitizing solution: Sensitizing solution is prepared using 0.02% w/v solution of sodium
thiosulfate.
3. Staining Solution: For staining, 0.2% solution of silver nitrate and 0.076% formalin in a
freshly prepared state is used.
4. Developing solution: It is prepared using 6% sodium carbonate, 0.0004% sodium thiosul-
fate, and 0.05% formalin. Here the order of mixing is important. Initially, sodium carbon-
ate is dissolved completely in a small amount of deionized water. The stipulated amount of
sodium thiosulfate is dissolved separately in deionized water, and finally both the solutions
are mixed with a subsequent addition of 35% formaldehyde and stored at room temperature
till further use.
5. Terminating solution: 12% acetic acid solution is prepared in deionized water.
6. Drying solution: 20% ethanol will serve as a drying solution in the final step.
�.7.�.4.1 Protocol for Performing Silver Staining Procedure
After completion of electrophoresis, the gel is removed from the cassette and soaked
in a sufficient volume of fixative solution for 2 h. The aim of fixation is to arrest the
protein movement from the gel matrix, thereby almost immobilizing the protein, and
also to remove the interfering ions and detergents from the gel. Prolonged fixation
also significantly improves the signal-to-noise ratio.
Fixation is followed by ethanol washing of the gel. Water should be avoided to
prevent swelling of the gel, and hence it becomes mandatory as well as preferential
to use ethanol as a wash reagent.
Washing is followed by sensitizing the solution to increase sensitivity and contrast
of staining to render it more differentiable.
The washing and sensitizing solutions are discarded and followed by cold silver
staining accompanied with rhythmic shaking to ensure uniform distribution of the
stain throughout the protein sample and to avoid unequal distribution leading to high
noise levels.
This step is followed by washing and the addition of developing solution to
develop the spots of proteins.
Once the desired intensity of bands is achieved, further reaction is stopped by add-
ing terminating solution to the gel immersed in the developing solution. Sensitivity
and detection limits of silver staining are very comparable to those obtained by mass
spectrometry; thereby this method was one of the most preferred techniques for pro-
tein visualization before mass spectrometric analysis.
�.7.�.4.2 Drawbacks of Silver Staining
Because the principle underlying silver staining is based on affinity of silver ions for
amino and carboxylic or allied functional groups in proteins, the method becomes
highly selective and specific. Proteins like mucoproteins, asialoglycoproteins, and
proteoglycans found in the mucous membranes of animals consist of high levels of
sulfated sugar fragments and are consequently poorly stained. The method is also not
suitable for quantitative protein expression analysis and lacks high throughput.
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