How sensitive is silver staining?
Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels. Classical Coomassie brilliant blue staining can usually detect a 50 ng protein band; silver staining increases the sensitivity typically 50 times.
Why is silver staining more sensitive?
Silver staining is the most sensitive colorimetric method for detecting total protein. The development process is essentially the same as for photographic film: silver ions are reduced to metallic silver, resulting in a brown-black color. Example gel stained with SilverXpress Silver Staining Kit.
Is silver stain more sensitive than Coomassie?
As a general protein stain, Coomassie is easier and more rapid; however, fluorescent and silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein.
Which protein staining method has the highest sensitivity?
silver staining
In terms of total protein detection, for example, silver staining is considered to be the most sensitive technique. However, the staining protocol is time-consuming and results are easily affected by a number of factors such as reagent quality, incubation times, and gel thickness [3].
What is the detection range of SDS PAGE?
It has a detection limit of ~ 0.1–0.5 μg protein, sensitive enough for most daily needs. Silver staining has greater sensitivity, but involves many more steps and solutions (see Silver Staining of SDS-polyacrylamide Gel).
Why silver staining is used in SDS-PAGE?
Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals.
What is silver staining in SDS-PAGE?
Silver staining is a powerful technique for protein identification in gels as silver binds to chemical sidechains of the amino acids, including the carboxyl and sulfhydryl groups. It was introduced in 1972 and later adapted for protein separation from the polyacrylamide gel electrophoresis.
How much protein do you need for silver staining?
100 ng protein for CBB R-250, 30-40 ng for G-250 and 5-10 ng for Silver staining.
When should I use silver stain?
The primary benefit of silver staining is its high sensitivity, as it is able to detect less than 1 ng of protein (Weiss et al. 2009), making it extremely useful for applications involving low protein levels.
What does silver stain stain?
Silver staining is the most sensitive colorimetric method for detecting total protein. The technique involves the deposition of metallic silver onto the surface of a gel at the locations of protein bands. Silver ions (from silver nitrate in the staining reagent) interact and bind with certain protein functional groups.
Does SDS PAGE denature proteins?
SDS is an amphipathic surfactant. It denatures proteins by binding to the protein chain with its hydrocarbon tail, exposing normally buried regions and coating the protein chain with surfactant molecules. For this reason, separation on a polyacrylamide gel in the presence of SDS occurs by mass alone.
Are there any disadvantages to using silver stain?
Another disadvantage of silver staining is that the use of formaldehyde when fixing the gel makes silver staining incompatible with mass spectrometry.
What does a silver stain on a protein mean?
Silver stain is not a linear stain with regard to protein amounts. A dark spot by silver does not mean a high concentration of protein, it only means the protein stains will by silver. Similarly, a faint band by silver does not mean low levels of protein, it only suggests a protein does not stain efficiently by silver.
How is silver staining used in mass spectrometric?
Here we describe a silver staining protocol that has been optimized for mass spectrometric analysis. The protocol results in confident protein identifications and high sequence coverage by MALDI MS and ESI MS due to a high recovery of peptides from the stained gel.
How is protein fixed in SDS PAGE stain?
The gel must be fixed by a non-modifying, precipitation procedure such at the ethanol (or methanol)-acetic acid method used here. If the protein is not fixed in the gel as a separate step from the staining, the protein will be washed away and your results will be compromised.