How do you make colloidal Coomassie?
first dissolve aluminum sulfate in Milli-Q water. thereafter add ethanol, homogenize, and mix CBB G-250 to the solution. as recently as the solution is completely dissolved, add phosphoric acid (the incorporation of the acid to the alcoholic media lets the Coomassie molecules aggregate into their colloidal state)
How do you Destain Coomassie Blue?
Destain the gel by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. If the gel still has a Coomassie Blue background then continue destaining until the background is nearly clear.
What is Coomassie staining for?
Description. 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.
Is Coomassie Blue reversible?
No. IB7710): Allows for complete, reversible staining of protein on nitrocellulose & PVDF membranes. Sensitivity is higher than Ponceau S (<10 ng of BSA in 10 mins as blue bands) and the staining is reversible in 5 minutes.
Can you 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.
Can you over Destain a gel?
As there are no fixative reagents in our destain- ing protocol, it is important that the gel is treated with acetic acid as part of the staining solution, at least once. Incomplete fixing of the proteins will result in a partial loss of signal. If this happens the gel can be restained.
What is the fastest way to Destain Coomassie gel?
FAST DESTAINING OF COOMASSIE GELS. Extract the gel from the sealing system (BioRad) alter running and introduce into a little tupperware with aprox 50ml of Coomassie solution (AcH : MeOH : H2O 10:45:45 with 0.25% Brilliant Blue G-250). Shake at RT and change de destaining solution until the bands can be observed.
Can you Western Blot after Coomassie?
The answer is yes: western blotting Coomassie-stained proteins can be done, but it’s not a simple or efficient process. As you know, there are two types of Coomassie stains – “classical” and “colloidal”. Proteins stained by one of these two methods will behave differently if you try to blot them afterwards.
Is Coomassie Blue toxic?
Harmful to aquatic life with long lasting effects. Material may be irritating to the mucous membranes and upper respiratory tract. May be harmful by inhalation, ingestion, or skin absorption. May cause eye, skin, or respiratory irritation.
Why do we stain with Coomassie 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.
Which is better G-250 or R-250 Coomassie dye?
Like R-250, Coomassie G-250 (also known as colloidal Coomassie dye) also offers relatively high sensitivity and involves a simple protocol. However, G-250 offers a faster staining protocol and eliminates the need for destaining the gel (you can easily visualize the protein bands against the light amber background).
How to dissolve Coomassie Brilliant Blue G-250 in water?
Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol and add 100 ml of 85% phosphoric acid while stirring continuously. When the dye has dissolved dilute to 1 l in water.
How does Coomassie blue dye bind to proteins?
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.
Which is the most common form of Coomassie dye?
Thermo Scientific Pierce Coomassie Brilliant Blue G-250 is one of the most common forms of coomassie dye, which is a key component of various colorimetric protein gel stains. Coomassie R-250 and G-250 dyes are two chemical forms of a disulfonated triphenylmethane compound that is commonly used as the basis of stains for detection…