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. Bio-Rad offers Coomassie stains in four formats.
Staining Protein Gels with Coomassie Blue. 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. Coomassie G-250 manifests a leuco form below pH 2.
Similarly, what amino acids does Coomassie blue bind to? In acidic conditions, the dye binds to proteins primarily through basic amino acids (primarily arginine, lysine and histidine), and the number of coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein.
Similarly, 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.
How does simply blue stain work?
Invitrogen™ SimplyBlue™ SafeStain is a ready-to-use, proprietary Coomassie™ G-250 stain that is specially-formulated for rapid, sensitive detection, and safe, non-hazardous disposal. This stain does not require the use of methanol or acetic acid, thus eliminating the need to dispose of hazardous waste.
What is silver staining used for?
Silver staining is the use of silver to selectively alter the appearance of a target in microscopy of histological sections; in temperature gradient gel electrophoresis; and in polyacrylamide gels.
Does Coomassie Blue stain DNA?
Protein Gel Stains Similar to DNA gels, there are several options for staining protein gels. The two most common stains are Coomassie Brilliant Blue and Silver stain. Coomassie is an anionic dye, which binds nonspecifically to proteins.
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.
How do you stain SDS gel?
Directions: Remove SDS-PAGE gel from glass and rinse once in ddH2O in a suitable container with a lid. Add enough Coomassie Stain to cover the gel by 1/2 inch (~ 1.5 cm). Microwave on high power for 40 seconds to 1 minute (until the Coomassie Stain boils).
How do you make Coomassie brilliant blue?
Coomassie Brilliant Blue solution. Dissolve 0.25 g of Coomassie Brilliant Blue R-250 in 90 ml of methanol:H2O (1:1, v/v) and 10 ml of glacial acetic acid. Filter the solution through a Whatman No. 1 filter to remove any particulate matter.
Which dye is used in SDS PAGE technique?
Once electrophoresis is complete, the gel can be stained using colored dyes such as Coomassie Brilliant Blue or ethidium bromide to make the separated proteins appear as distinct colored bands on the gel. Unbound dye is washed out from the gel.
Can you transfer Coomassie stained gel?
Use the copper stain if you plan to transfer the separated proteins to a membrane, as the Coomassie stain is not reversible. Only use the Coomassie stain on gels post-transfer to check the efficiency of the transfer, or if you have no plans to transfer and just want to observe the results of the SDS-PAGE separation.
What is protein staining?
Protein Gel Staining. Gel staining is an important visualization and detection step that follows protein polyacrylamide gel electrophoresis (PAGE), such as SDS-PAGE, native PAGE, or 2D-PAGE. Staining with silver is very sensitive, and can detect proteins in the low nanogram amounts.
What is Ponceau staining?
Ponceau S is a rapid and reversible stain for detecting protein bands on Western blot membranes and can be used with PVDF, nitrocellulose and cellulose acetate membranes*. Since, Ponceau-S staining is reversible, it allows further immunological detection.
Why and how are proteins fixed in the gel during staining?
After SDS-polyacrylamide gel electrophoresis proteins are “fixed” in the gel to prevent dispersion of the proteins and visualized by staining with a chromogenic dye. Acetic acid and methanol denature the protein and provide an acidic environment enhancing the interactions with dyes.
What is the role of bromophenol blue in SDS PAGE?
It is often used as a tracking dye during agarose or polyacrylamide gel electrophoresis. Bromophenol blue has a slight negative charge and will migrate the same direction as DNA, allowing the user to monitor the progress of molecules moving through the gel. The rate of migration varies with gel composition.
How does SDS PAGE separate proteins?
SDS-PAGE separates proteins primarily by mass because the ionic detergent SDS denatures and binds to proteins to make them uniformly negatively charged. Thus, when a current is applied, all SDS-bound proteins in a sample will migrate through the gel toward the positively charged electrode.
Is Coomassie Blue toxic?
Coomassie Briliant Blue R-250 Dye (6104-59-2) Potential Adverse human health effects and symptoms : Non-toxic if swallowed (LD50 oral, rat > 5000 mg/kg). Not irritant to skin. Not irritant to eyes. Symptoms/effects after inhalation : AFTER INHALATION OF DUST/MIST: Coughing.
What is the difference between Coomassie r250 and g250?
Between the two, Coomassie R-250 is the more commonly used variant for protein detection since it can be used to detect as little as 0.1 ug of protein. Like R-250, Coomassie G-250 (also known as colloidal Coomassie dye) also offers relatively high sensitivity and involves a simple protocol.