Why would someone use Ponceau S instead of AB HRP color development or Coomassie?
Ponceau S staining protocol takes about 20 minutes, is non-toxic, and a gentler solution than Coomassie Brilliant Blue. Ponceau S staining solution does not fix the protein, allowing for western blot analysis after staining, which is another key consideration.
What does a Ponceau stain show?
Ponceau S staining is a rapid and reversible staining method used for the detection of protein bands on Western blot membranes, Polyvinylidene fluoride (PVDF), nitrocellulose, and cellulose acetate membranes. The Ponceau S stain is reversible; this quality makes it useful for further immunological detection.
How do you stain a membrane with Coomassie?
BASIC PROTOCOL 2: COOMASSIE BLUE R-250 STAINING
- Place blot transfer membrane in a clear plastic box. Wash with water three times for 5 min each.
- Stain membrane with Coomassie blue stain for 5 min.
- Destain membrane with 50% methanol/7% acetic acid for 5 to 10 min.
- Rinse with water several times, then air dry.
Can Ponceau staining be used as loading control?
In conclusion, we have shown that reversible Ponceau S staining can be used advantageously over actin detection for quality or equal loading control in Western blotting. It is equally useful for this purpose, has similar or improved dynamic range, is extremely inexpensive, and takes no longer than 10 min.
Does Ponceau interfere with antibody binding?
There is no chance that your primary antibody binding be changed by doing Ponceau staining. Although the Ponceau stain should not block antibody binding, you can take a picture of the Ponceau stained membrane, and then remove the Ponceau by soaking in a large volume of water for a few minutes.
What is Coomassie stain used 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.
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.
Can you Western blot a Coomassie stained gel?
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.
What is Coomassie Blue made of?
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 of proteins in gel electrophoresis and Bradford-type assay reagents for protein quantitation.
How do you use Ponceau S staining solution?
To use Ponceau S (CST #59803); following electrotransfer onto membrane, briefly rinse the membrane in ddH2O, incubate the membrane in Ponceau S staining solution for five to ten minutes at room temperature, and then wash the membrane in ddH2O for one to five minutes, until red bands are visible.
What is the difference between Coomassie Brilliant Blue and Ponceau S?
Ponceau S staining protocol takes about 20 minutes, is non-toxic, and a gentler solution than Coomassie Brilliant Blue. Ponceau S staining solution does not fix the protein, allowing for western blot analysis after staining, which is another key consideration.
How to de-stain the membrane after dying with Ponceau S?
As with Coomassie, there is some background, but you can easily destain the membrane with water. Changing the solution several times reduces background; prolonged incubation destains the bands as well, returning the membrane to its white hue. But, you don’t need to de-stain the bands on the membrane completely after dying with Ponceau S.
What is Coomassie stain and how to use it?
The classic Coomassie stain consists of incubating the gel in a mix of methanol and acetic acid, which works as a solvent for the stain. But alcohol and acid treatment is harsh. It fixes the protein inside the gel, interfering with the transfer. If you pre-stain your gel, you will leave a lot of protein behind.