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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.
Why is Coomassie blue used in SDS-PAGE?
Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. This treatment allows the visualization of proteins as blue bands on a clear background.
Why do we use Coomassie stain in our procedure even though the buffer made the sample blue?
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.
Why is it necessary to stain the gel?
By coating the surface rather than seeping into the wood grain, a gel stain creates a professional-looking finish on even these most stubborn wood surfaces.
How does Coomassie blue staining work?
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.
How do you stain SDS PAGE with Coomassie Blue?
3. Stain: Dissolve 0.4g of Coomassie blue R350 in 200 mL of 40\% (v/v) HPLC grade methanol in water with stirring as needed. Filter the solution to remove any insoluble Page 2 material. Add 200mL of 20\% (v/v) acetic acid in water.
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 does Coomassie Blue stain bind to?
In acidic conditions, Coomassie dye primarily binds basic amino acids (arginine, lysine and histidine).
Why is Coomassie Blue a good stain for proteins?
The Coomassie Brilliant Blue dye which is commonly used in SDS-PAGE, was first described by the German scientist Volker Neuhoff. In the staining reaction, the Coomassie dye binds to proteins through ionic interactions between sulfonic acid groups and positive protein amine groups through Van der Waals attractions.
How do you stain SDS PAGE with Coomassie blue?
Coomassie R-250 Staining Protocol
- Prepare the staining solution containing 0.1\% Coomassie R-250 in 40\% ethanol, 10\% acetic acid.
- After electrophoresis, incubate 1 or 2 gels in a staining container containing 100 ml Coomassie Blue R-250 staining solution.
How do you stain Coomassie blue?
Stain: Dissolve 0.4g of Coomassie blue R350 in 200 mL of 40\% (v/v) methanol in water with stirring as needed. Filter the solution to remove any insoluble material. Add 200mL of 20\% (v/v) acetic acid in water. The final concentration is 0.1\% (w/v) Coomassie blue R350, 20\% (v/v) methanol, and 10\% (v/v) acetic acid.
How do you use Coomassie blue stain?
Coomassie Blue stain is used to stain the protein bands in polyacrylamide gels. One common way to use it is to dissolve the dye in a mixture of methanol, acetic acid, and water. This stain will permeate the gel, stain the protein, and also fix the protein in place.
What is Coomassie G250 stain used for?
There is another similar stain called Coomassie brilliant blue G-250, which is used in colloidal blue staining and for protein detection in the Bradford assay to determine concentration (see Quantification of Protein Concentration using UV absorbance and Coomassie Dyes ). SDS does not have to be removed from gels prior to staining.
Is Coomassie blue more sensitive than CBS?
However, there are other staining protocols more sensitive than CBS [1], [2]; and moreover, using Coomassie Blue dye can be time consuming. The time required to visualize proteins using CBS has been significantly reduced with fast staining protocols utilizing Coomassie Blue dye.
What is the staining step with Commassie dye?
Briefly, staining step with commassie or other dyes used for this purpose is to stain the protein bands. The gel also be stained, therefore you should de-stain the gel to reflect the protein bands with de-stained background.