What is the sensitivity of Coomassie blue?

Coomassie blue staining is a quick, simple, and affordable method for detecting proteins on gels. 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).

Is Coomassie blue or silver stain more sensitive?

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.

Is Coomassie more sensitive than Ponceau?

Coomassie brilliant blue R250 and amido black 10B are more sensitive than Ponceau S2. However with Coomassie brilliant blue R250 the background staining is high and also this stain is not removed easily.

What is the disadvantage of using Coomassie Blue stain?

A drawback to this stain can be the detection sensitivity, as Coomassie Blue binds to basic and hydrophobic amino acids; as a result, its sensitivity can vary somewhat, depending on the amino acid composition of the proteins being detected.

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 is Coomassie Brilliant Blue G-250?

Description. Thermo Scientific Pierce Coomassie Brilliant Blue G-250 is one of the most common forms of coomassie dye, which is a key component of various colorimetric protein gel stains. … Protein-binding causes the dye to change from reddish-brown to bright blue (absorption maximum equals 595 nm).

Does Coomassie stain all proteins?

As with all staining methods, Coomassie staining detects some proteins better than others, based on the chemistry of action and differences in protein composition. Thus, Coomassie staining can detect as little as 8–10 ng per band for some proteins and 25 ng per band for most proteins.

What is the most sensitive protein stain?

Silver staining 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.

What is the difference between silver stain and Coomassie?

Coomassie is much faster but 10-100XX less sensitive than silver stain. If your band is faint you should use silver, but its also a bit more tedious.

Can you stain membrane with Coomassie?

BASIC PROTOCOL 2: COOMASSIE BLUE R-250 STAINING Coomassie blue R-250 can be used with most types of blot membranes except nitrocellulose (high concentrations of organic solvents can dissolve nitrocellulose membranes).

How sensitive is a Western blot?

Results: Western blot sensitivity reached 100% in the samples studied and was thus superior to the sensitivities of serum analysis of venom-specific IgE using AlaSTAT microplate assay (90%) and skin tests (87%).

What is a Coomassie stain used for?

Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent (destaining). This treatment allows the visualization of proteins as blue bands on a clear background.

Can Coomassie Brilliant Blue interfere in SDS PAGE?

Having both dyes in a gel is a bad idea. CBB250 binds proteins and changes their mobility in SDS PAGE. The reason why the bromophenol blue dye front is smeared is usually due to high salt in the sample (greater than 0.1M) The BPB dye front should be straight as an arrow if the gel is run properly.

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 …

How does Coomassie Blue stain the proteins in the gel?

The Coomassie dyes (R-250 and G-250) bind to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions. Solutions of the dye, dark blue black at pH 7, turn a clear tan upon acidification. …

For what reason is Coomassie Blue a good stain for proteins?

Coomassie brilliant blue stain offers high sensitivity, low background, large linear range, and ease of use for the identification of proteins separated by gel. It is the most commonly used staining technique for the quantification of proteins after gel electrophoresis.

Which amino acids does Coomassie bind to?

In acidic conditions, Coomassie dye primarily binds basic amino acids (arginine, lysine and histidine).

What is colloidal Coomassie?

Bio-Rad’s QC colloidal Coomassie stain is a ready-to-use single-bottle protein stain that does not require the mixing of any components or addition of any alcohols. It is a special formulation of Coomassie G-250 that provides maximum sensitivity with low background for a wide variety of acrylamide gel chemistries.

What is the difference between R250 and G250 Coomassie Brilliant Blue G 250?

The more popular is Coomassie R250 (Reddish tinted blue) for electrophoresis (more sensitive: can detect as little as 0.1 µg of protein), and Coomassie G250 (Greenish tinted blue) for protein assay in solutions (because it is more convenient – soluble).

What is the difference between Coomassie Brilliant Blue G 250 and Coomassie brilliant blue R250?

Coomassie Brilliant Blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two methyl groups.

What is SDS PAGE?

SDS-PAGE is an electrophoresis method that allows protein separation by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel.

Why is it important to have a Coomassie stained gel of your samples when doing a Western?

Ponceau S Staining Solution and Coomassie Brilliant Blue Stain allow for the visualization of protein transfers after electrophoresis. They are important for confirming protein transfer and presence of the target of interest, saving time and valuable resources in your experiments.

Is Coomassie stain reversible?

Visualization of proteins in gels Use the copper stain if you plan to transfer the separated proteins to a membrane, as the Coomassie stain is not reversible.

Does Coomassie blue bind to all proteins?

The Bradford assay monitors a color change associated with the binding of Coomassie brilliant blue G-250 dye (CBBG) to protein in solution; the unbound dye absorbs at 470 nm, while the complex absorbs at 595 nm. Unfortunately, not all proteins bind to CBBG equally, because of differing amino acid compositions.

What is silver stain used for?

Silver stain techniques are widely used to detect nanogram quantities of proteins following electrophoresis. Silver nitrate is the silver source in most silver stain procedures. Silver staining is a highly sensitive method for detecting proteins in polyacrylamide slab gels.

Why is silver staining used?

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.

What are protein based stains?

Protein stains are stains caused by things that contain protein (go figure) including;

Why is SDS used in Western blotting?

SDS is generally used as a buffer (as well as in the gel) in order to give all proteins present a uniform negative charge, since proteins can be positively, negatively, or neutrally charged. … The gel electrophoresis step is included in western blot analysis to resolve the issue of the cross-reactivity of antibodies.

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.

Which of the following is good 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.