What does a Ponceau stain show?
Ponceau S is a rapid and reversible stain for detecting protein bands on Western blot membranes and can be used with PVDF, nitrocellulose and cellulose acetate membranes*.
What is the purpose of staining with Ponceau Red?
Ponceau S Staining Solution is supplied as ready to use. This product is recommended for rapid and reversible protein staining on nitrocellulose or PVDF membranes. This staining technique is often utilized to confirm protein electrotransfer in Western blotting assays prior to antibody-based detection.
Why do we stain with Ponceau S?
Ponceau S membrane staining is a nondestructive, reversible method to stain and detect proteins on nitrocellulose and PVDF membranes. The stain has minimal nonspecific interactions with the membrane surface and provides a reliable method for visualization of protein transfer to a membrane.
Does Ponceau interfere with antibody binding?
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.
How proteins are stained in western blotting?
Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored.
Is Ponceau stain fluorescent?
Even after thorough destaining, Ponceau stain can leave an autofluorescent residue on the membrane that increases background fluorescence.
What is Coomassie blue staining?
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.
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.
Why is Ponceau S used in Western blotting?
A Ponceau S stain is useful because it does not appear to have a deleterious effect on the sequencing of blotted polypeptides and is therefore one method of choice for locating polypeptides on western blots for blot-sequencing.
Can I Ponceau stain after blocking?
You should ponceau before blocking, as the presence of block on the membrane will increase background. To determine changes in protein expression you need to do western blot – it is specific for your protein, which ponceau is not. You can use ponceau to judge overall protein expression, but not for specific proteins.
What is total protein staining?
Total protein staining is a direct measure of the total amount of sample protein in each lane. For each lane, the sum of all the signal intensities of all the proteins in the lane is used for normalization. This more direct approach may increase the accuracy of normalization.
Can you Coomassie stain a membrane?
Yes, you can stain membranes with coomassie, but it isn’t reversible like ponceau S staining is. If you want to stain with coomassie, it is probably easier to do so in the gel.