Can you Coomassie stain a PVDF membrane?
Coomassie R-250: Stain PVDF membrane with 0.1% Coomassie R-250 in 40% MeOH for no longer than ONE MINUTE usually 15 to 20 seconds will suffice. (Staining for longer periods of time will result in high background and will interfere with extraction and cleavage)
What type of molecule does Coomassie Blue stain?
1 Coomassie Brilliant Blue staining. CBB R-250 and the dimethyl derivative CBB G-250 are disulfonated triphenylmethane dyes that stain protein bands bright blue.
Does Coomassie Blue stain all proteins?
The most common used protein stain is Coomassie Blue staining, which is based on the binding of Coomassie Brilliant Blue, which binds non-specifically to virtually all proteins.
How does Coomassie brilliant blue work?
Principle. The Coomassie Brilliant Blue G-250 dye has three forms: anionic (blue), neutral (green), and cationic (red). In an acidic environment, the red dye is converted into its blue form after binding to the protein of interest. If no protein binds to the dye, then the solution will remain brown.
How do you make Coomassie Blue stain?
To prepare 1 l of a staining solution, dissolve 1 g of Coomassie R250 to 300 ml of methanol. Then add 650 ml of MQ water and 50 ml of acetic acid. Stir the solution on a magnetic stirrer for 2 h. The solution can be filtered through a Whatman No.
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.
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 does Coomassie Blue stain proteins on a molecular level?
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.
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.
How long do you stain with Coomassie blue?
Stain for 1 hour at room temperature with gentle shaking. Bands will begin to develop within minutes.
What is Coomassie brilliant blue used for?
The Coomassie brilliant blue stain is used in the Bradford assay, a colorimetric protein assay, to quantify the proteins separated by gel. This assay is performed by determining the absorbance shift of the Coomassie Brilliant Blue G-250.
Can a Coomassie blue stain a nylon membrane?
Coomassie Blue R250 permanently stains membrane-bound proteins and is compatible with PVDF and nitrocellulose membranes, but it is incompatible with nylon membranes. This technique is relatively insensitive, with a detection limit of ~1.5 μg of protein.
What’s the best way to stain PVDF membrane?
Stain PVDF membrane with 0.1% Coomassie R-250 in 40% MeOH for no longer than ONE MINUTE usually 15 to 20 seconds will suffice. (Staining for longer periods of time will result in high background and will interfere with extraction and cleavage)
How are proteins stained with Coomassie Brilliant Blue?
Proteins are transblotted onto a polyvinylidene difluoride (PVDF) membrane and visualized by staining with Coomassie brilliant blue R-250, and a photograph of the protein pattern is taken.
Can a PVDF membrane be scanned for Edman sequencing?
The PVDF membrane can now be scanned, and protein bands submitted for N-terminal Edman sequencing. The PVDF membranes and buffers listed in the blotting protocol above generally works well for most proteins.