How does Coomassie Blue stain the proteins in the gel?

Staining Protein Gels with Coomassie Blue. 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. Coomassie G-250 manifests a leuco form below pH 2.

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.

Additionally, how do you Destain protein gel? Add fresh Destain solution to cover the gel by 3/4 inch (~ 2 cm). Tie Kimwipes in a simple knot and place 4 of them in the Destain solution around the gel. Try to avoid laying the Kimwipes on the gel as this will cause an uneven destaining. Microwave on high power for 40 seconds to 1 minute (until the Destain boils).

Consequently, what amino acids does Coomassie blue bind to?

In acidic conditions, the dye binds to proteins primarily through basic amino acids (primarily arginine, lysine and histidine), and the number of coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein.

How does a protein based gel develop?

In the first dimension, proteins are separated according to their isoelectric point (pI). To do so, the gel is applied to the top of an SDS-polyacrylamide slab. Electrophoresis is then applied horizontally across the top of the gel and the proteins migrate into the second-dimension gel.

Why and how are proteins fixed in the gel during staining?

After SDS-polyacrylamide gel electrophoresis proteins are “fixed” in the gel to prevent dispersion of the proteins and visualized by staining with a chromogenic dye. Acetic acid and methanol denature the protein and provide an acidic environment enhancing the interactions with dyes.

Does Coomassie Blue stain DNA?

Protein Gel Stains Similar to DNA gels, there are several options for staining protein gels. The two most common stains are Coomassie Brilliant Blue and Silver stain. Coomassie is an anionic dye, which binds nonspecifically to proteins.

What is silver staining used for?

Silver staining. Silver staining is the use of silver to selectively alter the appearance of a target in microscopy of histological sections; in temperature gradient gel electrophoresis; and in polyacrylamide gels.

Can you transfer Coomassie stained gel?

Use the copper stain if you plan to transfer the separated proteins to a membrane, as the Coomassie stain is not reversible. Only use the Coomassie stain on gels post-transfer to check the efficiency of the transfer, or if you have no plans to transfer and just want to observe the results of the SDS-PAGE separation.

How do you make Coomassie blue?

Dissolve 0.25 g of Coomassie Brilliant Blue R-250 in 90 ml of methanol:H2O (1:1, v/v) and 10 ml of glacial acetic acid. Filter the solution through a Whatman No. 1 filter to remove any particulate matter. Store at room temperature.

What is the role of bromophenol blue in SDS PAGE?

It is often used as a tracking dye during agarose or polyacrylamide gel electrophoresis. Bromophenol blue has a slight negative charge and will migrate the same direction as DNA, allowing the user to monitor the progress of molecules moving through the gel. The rate of migration varies with gel composition.

Is Coomassie blue light sensitive?

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).

Is Coomassie Blue toxic?

Coomassie Briliant Blue R-250 Dye (6104-59-2) Potential Adverse human health effects and symptoms : Non-toxic if swallowed (LD50 oral, rat > 5000 mg/kg). Not irritant to skin. Not irritant to eyes. Symptoms/effects after inhalation : AFTER INHALATION OF DUST/MIST: Coughing.

What stain is commonly used for proteins?

Coomassie Brilliant Blue

What happens to the absorbance of the dye Coomassie blue when it binds to protein?

When proteins bind to Coomassie blue in acid solution their positive charges suppress the protonation and a blue colour results. The binding of the dye to a protein causes a shift in the absorption maximum of the dye from 465 to 595 nm and it is the increase in absorbance at 595 nm that is monitored.

What is the difference between Coomassie r250 and g250?

Between the two, Coomassie R-250 is the more commonly used variant for protein detection since it can be used to detect as little as 0.1 ug of protein. Like R-250, Coomassie G-250 (also known as colloidal Coomassie dye) also offers relatively high sensitivity and involves a simple protocol.

How does SDS PAGE separate proteins?

SDS-PAGE separates proteins primarily by mass because the ionic detergent SDS denatures and binds to proteins to make them uniformly negatively charged. Thus, when a current is applied, all SDS-bound proteins in a sample will migrate through the gel toward the positively charged electrode.

Why is BSA used in Bradford assay?

BSA is used because of its stability to increase signal in assays, its lack of effect in many biochemical reactions, and its low cost, since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.

How does instant blue work?

The more InstantBlue, the more Coomassie dye that is free to bind to protein. Once the Coomassie dye is exhausted, then it can’t stain anymore protein. If that is ever the case, just simply add more stain and more dye will be bound.