How does Coomassie stain gel stain?
In acidic conditions, Coomassie dye binds to basic and hydrophobic residues of proteins, changing in color from a dull reddish-brown to intense blue. Thus, Coomassie staining can detect as little as 8–10 ng per band for some proteins and 25 ng per band for most proteins.
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. …
What did the results of the native and SDS-PAGE gels tell you about Fumarase?
It tells you that fumarase has a very little negative charge under conditions you run your native PAGE. And it also means that the real molecular mass of your protein is around 50 kDa.
What does Coomassie Blue stain bind to?
In acidic conditions, Coomassie dye primarily binds basic amino acids (arginine, lysine and histidine).
What is the difference between SDS-PAGE and native PAGE?
The major difference between native PAGE and SDS-PAGE is that in native PAGE, the protein migration rate is dependent on both the mass and structure, whereas in SDS-PAGE, the migration rate is determined only by protein’s mass. In native PAGE, protein samples are prepared in a non-denaturing and non-reducing buffer.
How much protein should I load on a Coomassie gel?
Ideally, it is best to load ≤2 µg per well of a purified protein or ≤20 µg of a complex mixture like whole cell lysates if you are doing Coomassie stain only.
How does Coomassie stain proteins on a SDS PAGE gel?
Samples were separated on a NuPAGE 4-12% Bis-Tris gel and stained with SimplyBlue SafeStain. In acidic conditions, Coomassie dye binds to basic and hydrophobic residues of proteins, changing in color from a dull reddish-brown to intense blue.
How does Native PAGE separate proteins?
In native-PAGE, proteins are separated according to the net charge, size, and shape of their native structure. Electrophoretic migration occurs because most proteins carry a net negative charge in alkaline running buffers. Thus native-PAGE separates proteins based upon both their charge and mass.
What does agarose gel analyze?
Electrophoresis through agarose or polyacrylamide gels is used to separate, analyze, identify, and purify DNA fragments. The technique is simple, rapid to perform, and capable of resolving fragments of DNA that cannot be separated adequately by other procedures, such as density gradient centrifugation.
Which amino acid does Coomassie bind to?
How long does it take to stain Coomassie gel?
Standard protocol – Coomassie Blue R-250. Gel may be prefixed in 50% MeOH, 10% HoAC, 40% H2O for 30 minutes to overnight. Stain gel in the above solution, with 0.25% Coomassie Blue R-250, for 2 – 4 hours, until the gel is a uniform blue color. Staining is complete when the gel is no longer visible in the dye solution.
What kind of dye is used for Coomassie staining?
A coomassie stained gel. Coomassie dyes (also known as Coomassie Brilliant Dyes) are a family of dyes commonly used to stain proteins in sodium dodecyl sulfate and blue native polyacrylamide gel electrophoresis (SDS-PAGE and BN-PAGE, respectively) gels. The gels are soaked in dye and excess stain is then eluted with a solvent (“destaining”).
Can you use methanol to de stain Coomassie?
The gel can be analyzed directly in the stain. Alternatively, a water or methanol:acetic acid de-staining step can be used to wash away excess unbound dye from the gel matrix. To expedite the staining process microwaves can be used with Coomassie stains not requiring methanol or acetic acid.
How are proteins recovered from a Coomassie stain?
Because no chemical modification occurs, excised protein bands can be completely de-stained and the proteins recovered for analysis by mass spectrometry or sequencing. We offer several Coomassie based protein stains that are easy-to-use and provides a simple visualization of proteins resolved by electrophoresis.