Why is silver staining more sensitive?

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.

Why is silver staining more sensitive than Coomassie?

Coomassie Blue staining is approximately 50-fold less sensitive than silver staining, however due to its simplicity binding Coomassie Blue is preferred. Staining methods that allow destaining of gels makes them more appealing, as destaining may be needed for certain downstream applications.

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].

When should I use silver stain?

Genomic and proteomic analysis. Silver staining is used to stain gels. The silver stain of proteins in Agarose gels was developed in 1973 by Kerenyi and Gallyas. Later it was adapted to polyacrylamide gels used in SDS-PAGE, and also for staining DNA or RNA.

Is silver staining more sensitive than Coomassie?

Silver staining is a more sensitive method of protein staining than Coomassie blue staining. Gels were washed in 45% aq. methanol 10% aq. acetic acid for 30 min, followed by 5% methanol 7% acetic acid for 30 min.

Is Coomassie blue reversible?

Sensitivity is higher than Ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes.

Is silver stain quantitative?

Silver staining has a narrow linear dynamic range (the range at which the level of staining is linear to the concentration), making it less suitable for quantification.

Which staining reagent is used for staining proteins?

The most common method of in-gel protein detection is staining with Coomassie dye. These stains either use the G-250 (“colloidal”) or the R-250 form of the dye. Colloidal Coomassie stains can be formulated to effectively stain proteins within 1 hour and requires only water (no methanol or acetic acid) for destaining.

What does ethidium bromide stain?

The most commonly used stain for detecting DNA/RNA is ethidium bromide. Ethidium bromide is a DNA interchelator, inserting itself into the spaces between the base pairs of the double helix. Ethidium bromide is a sensitive, easy stain for DNA. It yields low background and a detection limit of 1-5 ng /band.

Why is silver staining used?

Why does Coomassie Blue stain?

Coomassie Blue stain is used to stain the protein bands in polyacrylamide gels. The dye binds more tightly to the proteins than the to the gel matrix, however, so the dye can subsequently be removed from only the protein-free parts of the gel using a similar solvent from which the dye is omitted. This is the destain.

What amino acid does Coomassie Blue react with to result in a blue color change?

Under acidic conditions, the dye reacts primarily with arginine and to a lesser extent with lysine, histidine, tyrosine, tryptophan, and phenylalanine residues in proteins, producing a blue color with an absorbance maximum at 595 nm (the absorption range is between 575 nm and 615 nm), and 0.2–20 μg of protein can be …

Which is more sensitive, silver or zinc staining?

Zinc staining is as sensitive as typical silver staining (detects less than 1 ng of protein), and no fixation steps are required. Furthermore, the stain is easily removed, making this method compatible with MS or western blotting. Example gel stained with a zinc stain.

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.

How is silver staining used in protein detection?

What is silver staining? Silver staining is a special yet powerful staining technique that is used for the detection and identification of proteins in gels. This is because silver binds to the chemical terminal or side chains of amino groups i.e carboxyl and sulfhydryl groups.

How long does it take to sensitize silver stain?

Sensitize the gel using a tetrathionate sensitizing solution for 45 minutes. Rinse the gel with 20% ethanol in two-part (twice), at least 10 minutes for each wash. Rinse the gel four times with water, 10 minutes for each wash.