How do you Destain Coomassie gel for mass spec?
Destaining Solution: 25 mM ammonium bicarbonate in 50% acetonitrile. Mix 80 mg of ammonium bicarbonate with 20 ml of acetonitrile and 20 ml of ultrapure water. Store this solution at 4°C for up to 2 months.
How do you use Coomassie Blue stain?
Microwave for ~45 sec until the solution just starts to boil. Incubate at room temp with gentle shaking for 10-15 min. Heating allows the gel to stain faster. Alternatively, soak gel in stain for 1 hr at room temperature.
How long do you stain with Coomassie Blue?
Discard each rinse. Stain the mini-gel with enough Invitrogen™ SimplyBlue™ SafeStain (20-100 ml) to cover the gel. Stain for 1 hour at room temperature with gentle shaking. Bands will begin to develop within minutes.
How do you stain SDS-PAGE with Coomassie Blue?
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.
What does Coomassie blue stain tell you?
Popular Answers (1) 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.
How does Coomassie Brilliant Blue work?
Unbound Coomassie Blue absorbs light maximally at a wavelength of 465 nm, while the absorption maximum is at 595 nm when the dye is bound to protein. The Coomassie Brilliant Blue dye is best detected by using the 470nm excitation LED, and UV High filter for emission (Instead of the traditional lower white light).
Can you leave Coomassie stain overnight?
Coomassie blue staining of proteins Submerge the gel in the Commassie blue stain solution. Use just enough to completely submerge the polyacrylamide gel. Stain for 1-4 hours or overnight at room temperature with gentle shake. Coomassie blue stain solution can be reused for serveral times.
What does Coomassie Blue stain tell you?
What is Coomassie Blue made of?
Modern formulations typically use a colloid of the G form of dye in a solution containing phosphoric acid, ethanol (or methanol) and ammonium sulfate (or aluminium sulfate). The Bradford assay uses the spectral properties of Coomassie brilliant blue G-250 to estimate the amount of protein in a solution.
What is the Coomassie stain protocol?
Coomassie Stain Protocol Coomassie Stain Protocol The gel must be fixed prior to staining by a non-modifying, precipitation procedure such as the ethanol (or methanol)-acetic acid method used here. If the protein is not fixed in the gel as a separate step from the staining, the protein will be washed away and your results will be compromised.
How long does it take for Coomassie blue to stain?
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. Prior to complete staining, the gel will appear as a lighter area against the dark staining solution.
How to use Coomassie Blue G-250 for staining gel electrophoresis?
Protocol for Staining Gels with Coomassie Blue G-250. 1. Remove the gel from the electrophoresis chamber and place enough 0.5% Coomassie Blue G-250 (prepared in 50% methanol/ 10% acetic acid) to cover the gel.
How do you make Coomassie R250 staining solution?
Coomassie R250 staining solution (0,1 % Coomassie Blue R250 (w/w), 30 % methanol, 5 % acetic acid) 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.
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